A model of biofilm detachment

A general mathematical framework for modeling biofilm detachment is presented. The approach is founded on a material balance on biomass that equates the detachment rate to the product of a detachment frequency and a detaching particle mass. The model provides a theoretical basis for deriving many of the empirical detachment rate expressions in common use and can thus lend some insight into their physical and biological significance. By allowing for variation in the detachment frequency with depth in the biofilm, the model permits derivation of detachment expressions that reflect a dependence on chemical or physiological gradients in the biofilm. Analysis of literature data sets from two different biofilm systems suggests, in both cases, that detachment is a growth-associated phenomenon. (c) 1993 John Wiley & Sons, Inc.     

Development and characterisation of a novel three-dimensional inter-kingdom wound biofilm model

Chronic diabetic foot ulcers are frequently colonised and infected by polymicrobial biofilms that ultimately prevent healing. This study aimed to create a novel in vitro inter-kingdom wound biofilm model on complex hydrogel-based cellulose substrata to test commonly used topical wound treatments. Inter-kingdom triadic biofilms composed of Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus were shown to be quantitatively greater in this model compared to a simple substratum when assessed by conventional culture, metabolic dye and live dead qPCR. These biofilms were both structurally complex and compositionally dynamic in response to topical therapy, so when treated with either chlorhexidine or povidone iodine, principal component analysis revealed that the 3-D cellulose model was minimally impacted compared to the simple substratum model. This study highlights the importance of biofilm substratum and inclusion of relevant polymicrobial and inter-kingdom components, as these impact penetration and efficacy of topical antiseptics.     

A novel in vitro flat‐bed perfusion biofilm model for determining the potential antimicrobial efficacy of topical wound treatments

Aims: To develop an in vitro flat‐bed perfusion biofilm model that could be used to determine the antimicrobial efficacy of topically applied treatments. Methods and Results: Pseudomonas aeruginosa and Staphylococcus aureus biofilms were grown within continuously perfused cellulose matrices. Enumeration of the biofilm density and eluate was performed at various sampling times, enabling determination of the biofilm growth rate. Two antimicrobial wound dressings were applied to the surface of mature biofilms and periodically sampled. To enable real‐time imaging of biofilm growth and potential antimicrobial kinetics, a bioluminescent Ps. aeruginosa biofilm was monitored using low‐light photometry. Target species produced reproducible steady‐state biofilms at a density of c. 10⁷ per biofilm support matrix, after 24‐h perfusion. Test dressings elicited significant antimicrobial effects, producing differing kill kinetic profiles. There was a good correlation between photon and viable count data. Conclusions: The model enables determination of the antimicrobial profile of topically applied treatments against target species biofilms, accurately differentiating bactericidal from bacteriostatic effects. Moreover, these effects could be monitored in real time using bioluminescence. Significance and Impact of the Study: This is the first in vitro biofilm model which can assess the antimicrobial potential of topical therapies in a dynamic growth environment.     

Modelling antisepsis using defined populations of facultative and anaerobic wound pathogens grown in a basally perfused biofilm model

An in vitro model was developed to assess the effects of topical antimicrobials on taxonomically defined wound biofilms. Biofilms were exposed over seven days to povidone-iodine, silver acetate or polyhexamethylene biguanide (PHMB) at concentrations used in wound dressings. The rank order of tolerance in multi-species biofilms, based on an analysis of the average bacterial counts over time was P. aeruginosa > methicillin-resistant Staphylococcus aureus (MRSA) > B. fragilis > S. pyogenes. The rank order of effectiveness for the antimicrobials in the biofilm model was povidone-iodine > PHMB > silver acetate. None of the test compounds eradicated P. aeruginosa or MRSA from the biofilms although all compounds except silver acetate eliminated S. pyogenes. Antimicrobial effectiveness against bacteria grown in multi-species biofilms did not correlate with planktonic susceptibility. Defined biofilm populations of mixed-species wound pathogens could be maintained in the basal perfusion model, facilitating the efficacy testing of treatments regimens and potential dressings against multi-species biofilms composed of wound isolates.     

A porcine model of skin wound infected with a polybacterial biofilm

A clinically relevant porcine model of a biofilm-infected wound was established in 10 minipigs. The wounds of six experimental animals were infected with a modified polymicrobial Lubbock chronic wound biofilm consisting of Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Bacillus subtilis. Four animals served as uninfected controls. The wounds were monitored until they had healed for 24 days. The biofilm persisted in the wounds up to day 14 and significantly affected healing. The control to infected healed wound area ratios were: 45%/21%, 66%/37%, and 90%/57% on days 7, 10 and 14, respectively. The implanted biofilm prolonged inflammation, increased necrosis, delayed granulation and impaired development of the extracellular matrix as seen in histological and gene expression analyses. This model provides a therapeutic one-week window for testing of anti-biofilm treatments and for research on the pathogenesis of wound infections in pig that is clinically the most relevant animal wound healing model.     

In vitro biofilm models to study dental caries: a systematic review

The aim of this systematic review is to characterize and discuss key methodological aspects of in vitro biofilm models for caries-related research and to verify the reproducibility and dose-response of models considering the response to anti-caries and/or antimicrobial substances. Inclusion criteria were divided into Part I (PI): an in vitro biofilm model that produces a cariogenic biofilm and/or caries-like lesions and allows pH fluctuations; and Part II (PII): models showing an effect of anti-caries and/or antimicrobial substances. Within PI, 72.9% consisted of dynamic biofilm models, while 27.1% consisted of batch models. Within PII, 75.5% corresponded to dynamic models, whereas 24.5% corresponded to batch models. Respectively, 20.4 and 14.3% of the studies reported dose-response validations and reproducibility, and 32.7% were classified as having a high risk of bias. Several in vitro biofilm models are available for caries-related research; however, most models lack validation by dose-response and reproducibility experiments for each proposed protocol.     

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.     

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.     

In vitro flowering of orchids

Abstract

Flowering is the most elusive and fascinating of all plant developmental processes. The ability to induce flowering in vitro in orchids would reduce the relatively long juvenile phase and provide deeper insight into the physiological, genetic and molecular aspects of flowering. This review synthesizes all available studies that have been conducted on in vitro flowering of orchids with the objective of providing valuable clues as to the mechanism(s) that is possibly taking place.     

In vitro mammalian mutagenesis as a model for genetic lesions in human cancer

Recently in vitro assays of mutagenesis have been criticized as being poorly predictive of long-term in vivo rodent assays of carcinogenicity. Questions have also been raised concerning the relevance of rodent assays to human risk. In vitro assays using mammalian cells can detect most types of genetic lesions thought to be important in human malignant disease. Molecular and cytogenetic analyses of mutations induced by a variety of genotoxic compounds at the heterozygous thymidine kinase locus in mouse lymphoma cells indicate that this in vitro assay does indeed register the range of genetic lesions recently found in a wide variety of human tumors. The types and complexity of the induced lesions are reflected in mutant colony phenotype in a compound-specific fashion. These studies point to the use of appropriate in vitro mammalian mutagenesis assays as new model systems for dissecting the genetic lesions important in human carcinogenesis, and as a means of determining the potential for compounds to induce such lesions.     

Optical method for long-term and large-scale monitoring of spatial biofilm development

A method was developed that allows biofilm monitoring on the square centimeter scale over extended periods of time. The method is based on image acquisition using a desktop scanner and subsequent image analysis. It was shown that results from grey level analysis are highly correlated with physical properties of the biofilm like average biomass and biofilm thickness. The scanner method was applied to monitor overall biofilm growth, detachment, and surface roughness during two 3 and 4 week long experiments. Two significantly different growth dynamics during the biofilm development could be identified, depending on the biofilm history. Surface roughness on transects in flow direction was always higher than on transects perpendicular to the flow, reflecting the anisotropic characteristics of biofilms growing in a flow field.     

In vitro method to assess the antimicrobial activity and potential efficacy of novel types of wound dressings

AIMS: To develop a simple, reproducible in vitro static diffusion method using cellulose disks and defined species to test antimicrobial efficacy of wound dressings. METHODS AND RESULTS: Cellulose disks were inoculated by immersion in cell suspensions of target species Staphylococcus epidermidis, Candida albicans and Fusobacterium nucleatum. Test and control wound dressings were cut into equal sized squares (25 x 25 mm) and applied to the surface of 10-mm thick tryptone yeast extract agar on test beds. Following a 2-h equilibration period, inoculated cellulose disks were inserted (one per dressing) at the interface between dressing and agar surface and a small weight applied over each square. At various sampling times, disks were removed and surviving cells enumerated by viable counts. Disk to disk variation for microbial loading was assessed using S. epidermidis for both initial (n = 16) and standard treatment (n = 16) conditions. The coefficient of variation was low (<5%) indicating good reproducibility for cell loading and treatment position on the test bed. Replicate assays (n = 6) using S. epidermidis and oxyzyme gels produced similar kill rates with low scatter (R2 > 0.9) indicating good reproducibility between assays. Significant differences (P < 0.05) in kill rates were observed for different target species, types of dressing and test bed conditions (+/-blood and nutrients). CONCLUSIONS: The method is reproducible and useful in tracking the death kinetics of test species, enabling the comparison of different types of dressing. SIGNIFICANCE AND IMPACT OF THE STUDY: The reported method has significant advantages over established test procedures; it can be applied equally across a wide range of target species (including anaerobes and yeasts), a wide range of conditions, and different types of surface dressings, including those relying upon oxygen diffusion.     

In vitro effect of clarithromycin and alginate lyase against helicobacter pylori biofilm

It is now established that the gastric pathogen Helicobacter pylori has the ability to form biofilms in vitro as well as on the human gastric mucosa. The aim of this study is to evaluate the antimicrobial effects of Clarithromycin on H. pylori biofilm and to enhance the effects of this antibiotic by combining it with Alginate Lyase, an enzyme degrading the polysaccharides present in the extracellular polymeric matrix forming the biofilm. We evaluated the Clarithromycin minimum inhibition concentration (MIC) on in vitro preformed biofilm of a H. pylori. Then the synergic effect of Clarithromycin and Alginate Lyase treatment has been quantified by using the Fractional Inhibitory Concentration index, measured by checkerboard microdilution assay. To clarify the mechanisms behind the effectiveness of this antibiofilm therapeutic combination, we used Atomic Force Microscopy to analyze modifications of bacterial morphology, percentage of bacillary or coccoid shaped bacteria cells and to quantify biofilm properties. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1584–1591, 2016     

In vitro pollination as a model for studying fertilization in maize (Zea mays L.)

Summary In vitro pollination was conducted using excised segments of maize female spikelets to determine the effects of age and silk length on fertilization efficiency and developmental pattern. Ovary development after 15 days resulted in: (1) normal kernels, (2) abnormal kernels and (3) enlarged ovaries; the percentages of each class varied with age. Evidence of double fertilization was observed in both normal and abnormal kernels. In vitro fertilization was traced using silk excision and autoradiography with ³²P-radiolabelled pollen and occurred between 4 and 7 h after the pollination of 4.5-cm-long silks. This study supports the validity of the in vitro pollination method for studying fertilization and emphasizes the importance of using a developmentally sensitive index (silk length) for establishing female developmental stage.     

In vitro determination of generation times for Entodinium exiguum, Ophryoscolex purkynjei and Eudiplodinium maggii

ABSTRACT. Most previously reported generation times for rumen ciliate protozoa are longer than would be required to prevent their being flushed out of the rumen. In an earlier study from this lab, using a sequential transfer procedure, generation times between 12 and 13 h were determined for both Epidinium caudatum and Entodinium caudatum . This would permit these species to be maintained in a rumen with a fluid volume turnover rate as rapid as twice a day. In this study, generation times were estimated for Entodinium exiguum (13.2 h), Eudiplodinium maggii (26.8 h), and Ophryoscolex purkynjei (29 h), by sequential transfer at both 12 and 24 h time periods. The generation time for E. exiguum is lower than reported for this and other Entodinium species as determined by logarithmic growth from a small inoculum, but similar to that obtained for Ent. caudatum using sequential transfer. Eudiplodinium maggii and O. purkynjei generation times are similar to previous estimates of 24- and 24–48 h, respectively. However, it was observed that after an adaptation period of 36 to 48 h (generally 3–4 transfers) cell concentrations decreased and generation times were markedly decreased, i.e. 12.2 h for Ent. exiguum , 15.0 h for E. maggii and 12.8 h for O. purkynjei . In a separate study, varying both the concentration of Epidinium and the quantity of substrate fed per cell had no effect on generation time.     

Diagnosis of biofilm infections: current methods used,challenges and perspectives for the future

The diagnosis of biofilms continues to be a challenge, and there is no standardized protocol for such a diagnosis in clinical practice. In addition, some proposed methodologies are expensive to require significant amounts of time and a high number of trained staff, making them impracticable for clinical practice. In recent years, mass spectrophotometry/matrix-assisted laser desorption ionization time of flight (MALDI-TOF) has been applied it in biofilm studies. However, due to several problems and limitations of the technique, MALDI-TOF is far from being the gold standard for identifying biofilm formation. The omics analysis may prove to be a promising strategy for the diagnosis of biofilms in clinical laboratories since it allows the identification of pathogens in less time than needed for conventional techniques and in a more specific manner. However, omic tools are expensive and require qualified technical expertise, and an analysis of the data obtained needs to be careful not to neglect subpopulations in the biofilm. More studies must therefore be developed for creating a protocol that guarantees rapid biofilm identification, ensuring greater chances of success in infection control. This review discusses the current methods of microbial biofilm detection and future perspectives for its diagnosis in clinical practice.     

新生隐球菌生物膜动物感染模型的构建及其活性的检测

目的 构建一种简便易行的新生隐球菌生物膜感染动物模型.方法 采用大鼠皮下置管法构建新生隐球菌生物膜感染动物模型,并使用电子扫描显微镜观察新生隐球菌体内形成生物膜的结构;采用MTT法对获得的体内生物膜结构进行活性的检测.结果 成功构建了新生隐球菌生物膜大鼠感染模型;MTT法检测体内生物膜活性表明,随着体内培养时间的延长,生物膜活性增强,与直接镜检和电镜观察结果一致.结论 新生隐球菌生物膜大鼠模型简单易行,便于操作,对于研究新生隐球菌生物膜体内活性的研究具有一定的实际应用价值.     

The diffusive vesicle supply center model for tip growth in fungal hyphae

We propose the diffusive vesicle supply center model for tip growth in fungal hyphae. The model is based on the three-dimensional vesicle supply center (VSC) model [Gierz, G., Bartnicki-García, S., 2001. A three-dimensional model of fungal morphogenesis based on the vesicle supply center concept: J. Theor. Biol. 208, 151-164], but incorporates two aspects of a more realistic vesicle delivery mechanism: vesicle diffusion from the VSC and a finite rate constant for vesicle fusion with the cell membrane. We develop a framework to describe tip growth for a general class of models based on the vesicle supply center concept. Combining this with a method for calculating the steady state distribution of diffusive vesicles we iteratively solve for stationary cell shapes. These show a blunter tip than predicted by the original VSC model, which we attribute to increased forward-directed vesicle delivery via diffusion. The predicted distance between the VSC and the utmost tip of the cell is set by the ratio between the diffusion constant and the rate constant for vesicle exocytosis. Combined with the cell radius, these define the only dimensionless parameter for our model.     

In vitro stimulation of human endothelial cells by derivatized dextrans

Summary Derivatized dextrans exert a stimulatory effect on the in vitro growth of human umbilical vein endothelial cells (HUVEC). Measurements of growth were monitored by [³H]thymidine uptake and cell numbers. Our results show that some derivatized dextrans at 4 μg/ml (88 nM) increase the [³H]thymidine incorporation, whereas starting dextran (40 000 Da), dextran sulfate, and carboxymethyl dextran have no effect. In addition, heparin under similar experimental conditions shows a slight inhibitory effect on the HUVEC growth. The stimulatory effect of derivatized dextrans was also found when HUVEC grew during 7 days in medium containing 2% fetal bovine serum. We also observed that derivatized dextrans had no effect on the mitogenic activity of acidic fibroblast growth factor, a mitogenic factor for several cell types including HUVEC. By assessment of [³H]thymidine uptake at 48 h without serum, we concluded that the exogenous growth factors were not involved in the proliferative activity of these components. The stimulatory effects are related to the chemical nature and the proportion of substituents on the synthetic polysaccharides. The data indicate that benzylamide sulfonated groups play a key role in the stimulation of HUVEC growth. Neither carboxyl nor sulfate groups alone exhibit this effect. Thus, the stimulatory capacity of dextran derivatives depends strongly on the respective ratios of the functional groups.