Name: porphyromonas gingivalis as a model organism for assessing interaction of anaerobic bacteria with host cells
Uploaded: 2015-12-17T13:00:00.000+00:00
Duration: 12 min 16 s
Description: Watch this Scientific Journal Video about Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells at JoVE.com

We would like to thank Dr. Hiroshi Miyazaki, Dr. Scott Henderson, Dr. Todd Kitten, Dr. Justin Hutcherson, Dr. Catherine Jauregui, and Collin R. Berry. This work was supported by NIH NIDCR grants R01DE016124, R01DE018039, and R01DE023304 to J.P. Lewis.
Microscopy was performed at the VCU Department of Anatomy and Neurobiology Microscopy Facility, supported, in part, with funding from NIH-NINDS Center core grant (5P30NS047463).

Authors have nothing to disclose.

Все вышеперечисленные методы могут быть использованы для разработки конкретных анализов для оценки взаимодействия анаэробных бактерий с эукариотических клетках. Тем не менее, есть несколько соображений, чтобы успешно выполнять эксперименты. Во-первых, являются микробные штаммы быт?...

Анаэробные бактерии колонизируют практически все поверхности человеческого тела. Хотя преобладает в флоры кишечной и мочеполовой путей, где концентрация кислорода низка, они также существуют на высоких уровнях на коже, во рту, носу, горле и 1. Анаэробные бактерии являются частой причиной эндогенных инфекций и часто изолированы от больных сайтов. Тем не менее, из-за их разборчивого природы, анаэробы может быть трудно выделить и культивировать. Исследования с участием анаэробных бактерий должно быть сделано в ограниченных условиях. Современные методы анаэробной-культуры позволяют исследователям имитировать настройки анаэробные, необходимые для изучения многих анаэробных лабораторных штаммов или клинических изолятов даже 2,3. Патогенные бактерии анаэробные разработала динамичные отношения и сотрудничество с эволюцией клеток-хозяев, в которых они проживают. Большинство анаэробов чувствительны к убийству принимающей иммунного ответа до достижения infectiленные уровни. Тем не менее, некоторые патогенные бактерии выработали механизмы эвакуации из или подорвать иммунного ответа. Они достичь этой цели с помощью таких механизмов, как уклонение от иммунного распознавания, обезвреживания иммунных медиаторов, изменения клеточного иммунитета, вторжения клеток-хозяев, и изменения иммунных сигнализации 4. Porphyromonas gingivalis, грамотрицательная анаэробные причастны как в устной и внеротовые заболевания, Одним из примеров весьма адаптированной бактериальным патогеном, способной вызвать патогенные изменения в организме хозяина 5-7. Карманы биопленки налета начисленные в глубоких трещинах, образующихся между зубами и десневой ткани слизистой оболочки может питать анаэробные бактерии, которые защищены от атмосферного кислорода 8. Эти зубодесневых карманов служить нишей для различных анаэробных патогенных микроорганизмов, таких как P. gingivalis 9. П. gingivalis является краеугольным камнем патоген, который способен переделыватьING устное микробное сообщество в целях содействия развитию и прогрессирования заболеваний пародонта 10. Это производит большое количество факторов вирулентности, которые активны в отношении широкого спектра хозяев, и белками обеспечивает механизмы для уклонения от защитных сил организма 11. Он также способен вторжения эпителиальные, эндотелиальные, фибробластов и клеток периодонтальной связки в пробирке 12-14 и в естественных условиях 15. Эффективно вторжения клеток-хозяев, Р. gingivalis может избежать иммунитет хозяина. Эффективное вторжение клетках-хозяевах позволяет не только бактерии, чтобы избежать защитные силы организма, но также служит в качестве резервуара для будущего повторной инфекции, а также изменяет клетки-хозяина. Исследования молекулярных механизмов, участвующих в адгезии и интернализации бактерии клетками-хозяевами необходимы. Исследования в нескольких лабораториях ориентирована на понимание молекулярных событий, связанных с интернализации P. gingivalis клетками-хозяевамиа также механизмов, используемых для подавления и захватить иммунный ответ и выжить враждебных защитными механизмами. Есть много анализы, способные идентификации и характеристике патогенов, которые способны вторжения клеток-хозяев. Тем не менее, исследования в пробирке с анаэробных патогенов представляют многие экспериментальные проблемы для исследователя главным образом, потому что это трудно выполнить исследования, которые полагаются на громоздких инструментов в отсутствие кислорода. Это усугубляется тем, что эукариотические клетки требуют кислорода для роста и, следовательно, должны быть получены отдельно в культуре ткани инкубатора. Один из способов избежать таких препятствий будет проводить исследования под атмосферным кислородом, но, что бы сделать рост анаэробных бактерий невозможно. Другой способ заключается в использовании тепла убитых бактерий, чтобы заразить и изучить хост-клеточные взаимодействия. Тем не менее, существуют различия между тепловыми убитых и жизнеспособных бактерий, которые уменьшают релевантность хозяин-патоген interacti16. Это центральный учиться жизнеспособные бактерии с неизмененной выражения, взаимодействующего с клетками-хозяевами; Следовательно, способы культивирования P. для gingivalis в обстановке анаэробного даны. Кроме того, две простые рентабельные протоколы продемонстрировано оценки способности P. gingivalis быть усвоены пупочной вены человека эндотелиальных клеток (HUVECs). Первый протокол основан на популярной антибиотиками анализа защиты. Хотя анализ прост, соображения при использовании анаэробных микроорганизмов приведены. Второй протокол требует использования флуоресцентного сканирующего микроскопа визуализировать взаимодействие и внутреннюю P. gingivalis. Каждый метод имеет свои ограничения и преимущества, которые будут обсуждаться, чтобы обеспечить исследователю план для изучения инвазивность анаэробных бактерий. Хотя в настоящее время рукопись изучает Р. gingivalis и HUVECs, эти протоколы могут быть использованы для многих других анаэробных бактерий, а такжеа для других типов клеток-хозяев.

<table><tbody><tr><td>Vinyl Anaerobic Chamber-Type B</td><td>Coy Laboratory Products</td><td></td><td>Model 2000 incubator&amp;nbsp;</td></tr><tr><td>TSA II Trypticase Soy Agar with 5% Sheep Blood</td><td>BBL</td><td>221261</td><td></td></tr><tr><td>Human Umbilical Vein Endothelial Cells 10-donor Pool</td><td>LifeLine Technology</td><td>FC-0044</td><td></td></tr><tr><td>VascuLife VEGF Medium Complete Kit</td><td>LifeLine Technology</td><td>LL-0003</td><td></td></tr><tr><td>TrypKit</td><td>LifeLine</td><td>LL-0013</td><td></td></tr><tr><td>Saponin</td><td>Riedel-de Haen</td><td>16109</td><td></td></tr><tr><td>Gentamicin Sulfate Salt</td><td>Sigma-Aldrich</td><td>G-1264</td><td></td></tr><tr><td>Metronidazole</td><td>Sigma-Aldrich</td><td>M-3761</td><td></td></tr><tr><td>BCECF-AM</td><td>LifeTechnologies</td><td>B1150</td><td></td></tr><tr><td>TRITC Phalloidin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P1951</td><td></td></tr><tr><td>18 mm Circular Coverslips</td><td>Electron Microscopy Sciences</td><td>72222-01</td><td></td></tr><tr><td>VectaShield Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr></tbody></table>

porphyromonas gingivalis as a model organism for assessing interaction of anaerobic bacteria with host cells

Анаэробные бактерии гораздо больше, чем аэробов во многих нишах человека, таких как кишечника, полости рта, влагалища и. Кроме того, анаэробные инфекции являются общими и часто из числа коренного населения. Способность некоторых анаэробных патогенных микроорганизмов к вторжению клетки человека дает им адаптивные меры побега врожденный иммунитет, а также модулируют поведение клеток-хозяев. Однако, убедившись, что анаэробные бактерии живут при экспериментальном исследовании событий может создать проблемы. Porphyromonas gingivalis, грамотрицательные анаэробы, способен вторжения различные эукариотических без фагоцитирующих клеток. В этой статье описывается, как успешно культура и оценить способность P. gingivalis вторгнуться пупочной вены человека эндотелиальных клеток (HUVECs). Были разработаны два протокола: один для измерения бактерии, которые могут успешно вторжения и выжить в хосте, а другой для визуализации бактерий, взаимодействующих с клетками-хозяевами. Эти методы требуют использования в anaeРобик камера для подачи P. gingivalis с анаэробной среде для оптимального роста. Первый протокол основан на анализе антибактериальной защиты, которая в значительной степени используется для изучения вторжение клеток-хозяев бактериями. Тем не менее, антибиотик анализ защита ограничена; только внутриклеточные бактерии, которые культивируемых после лечения антибиотиками и клетки-хозяина лизиса измерить. Для оценки всех бактерий, взаимодействующих с клетками-хозяевами, и живых и мертвых, мы разработали протокол, который использует флуоресцентной микроскопии для изучения хозяин-патоген взаимодействия. Бактерии флуоресцентно меченных с 2 &#39;, 7&#39;-бис- (2-карбоксиэтил) -5- (и-6) -carboxyfluorescein ацетоксиметил эфир (BCECF-AM) и использовали для инфицирования эукариотических клеток в анаэробных условиях. После фиксации с параформальдегидом и проницаемости с 0,2% Triton X-100, клетки-хозяева помечены TRITC фаллоидином и DAPI для обозначения клеток цитоскелета и ядра, соответственно. Несколько ИМАГЭС, принятые на различных координационных центров (Z-стека) получены для височно-пространственной визуализации бактерий. Методы, используемые в данном исследовании, могут быть применены к любому обрабатываемой анаэробной и любой эукариотической типа клеток.

Протоколы, описанные выше, были использованы в изучении хозяин-патоген взаимодействия между P. gingivalis и эндотелиальные клетки. П. gingivalis W83 и П. gingivalis V3150 неся удаление PG0228 были использованы в исследовании. PG0228 прогнозируется кодирует белок, который может изменить уровни РНК ...

Watch this Scientific Journal Video about Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells at JoVE.com

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis в качестве модельного организма для оценки интерактивности анаэробных бактерий с клеток-хозяев

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

porphyromonas-gingivalis-as-model-organism-for-assessing-interaction

Research

JoVE Journal

Immunology and Infection

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

26.2K Views.  Virginia Commonwealth University. This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type. 

Video: Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

Оценка жизнеспособности синтетических бактерий консорциума на интерфейсе в Vitro хост микробов кишечника

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Biochemistry

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Мышь Модель для патогенных микроорганизмов, вызванной хроническим воспалением на локальном и системном сайтов

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

Надежная Лигатура индуцированной модели Мурин пародонтит для оценки устных нейтрофилов

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

Модели вагинальной колонизации мышей анаэробно выращенными бактериями

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While in-house grown 2D epithelial monolayers lack true representation of the in vivo situation, commercially available tissue models are often overlooked due to their cost and practicality. However, with careful planning, such models provide reproducible platforms for a vast array of different applications. Here, we report the use of epithelial models that can be utilized for a wide variety of experimental purposes to investigate host-pathogen interactions in various ecological niches, such as the oral cavity, skin, and vaginal mucosa. From simple planktonic cells to complex biofilm co-culture, epithelial models are used to assess microbial adherence and invasion, and to evaluate the host response at a transcriptional and/or protein level, with scope for more detailed profiling using different omics approaches. Furthermore, these biological systems can be used as more accurate test beds for evaluating conventional and novel antimicrobial activity in a complex host-pathogen microenvironment in vitro. The protocols described herein document how models are handled upon arrival and prepared in the laboratory for co-culture stimulation with biofilm communities. The methods detail how experimental outputs are achieved from the model systems, including the processing of tissue, the co-culture setup, and data generation. These experiments include host gene expression through single- and multiplex qPCR analyses and inflammatory protein detection using ELISAs. In conclusion, epithelial models provide useful in vitro systems for preclinical investigatory studies into simple or complex host-pathogen interactions.

The present protocol outlines the application of organotypic tissue models for investigating host-pathogen interactions in vitro. Specifically, the model system described uses a multi-layered oral epithelium exposed to microorganisms associated with biofilms (dental plaque) within the context of oral health versus disease.

Органотипические модельные системы тканей для исследования взаимодействий хозяина и патогена in vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While ...

Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial diversity on earth. Novel approaches are required to culture these uncultured bacteria so that investigators can effectively study their physiology and lifestyle using the powerful tools available in the laboratory. The Candidate Phyla Radiation (CPR) is one of the largest groups of uncultivated bacteria, comprising ~15% of the living diversity on earth. The first isolate of this group was a member of the Saccharibacteria phylum, 'Nanosynbacter lyticus' strain TM7x. TM7x is an unusually small bacterium that lives as a symbiont in direct contact with a bacterial host, Schaalia odontolytica, strain XH001. Taking advantage of the unusually small cell size and its lifestyle as a symbiotic organism, we developed a protocol to rapidly culture Saccharibacteria from dental plaque. This protocol will show how to filter a suspension of dental plaque through a 0.2 &#181;m filter, then concentrate the collected Saccharibacteria cells and infect a culture of host organisms. The resulting coculture can be passaged as any normal bacterial culture and infection is confirmed by PCR. The resulting binary culture can be maintained in the laboratory and used for future experiments. While contamination is a possibility, the binary culture can be purified by either further filtering and reinfection of host, or by plating the binary culture and screening for infected colonies. We hope this protocol can be expanded to other sample types and environments, leading to the cultivation of many more species in the CPR.

We demonstrate a method for isolating difficult-to-grow members of the novel bacterial phylum, Saccharibacteria, by filtering dental plaque and co-culturing with host bacteria.

Создание стабильных бинарных культур симбиотических сахариобактерий из полости рта

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial ...

Biology

Measuring the Migration and Biofilm Formation of Various Bacteria

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923&#160;and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

<meta charset="utf8"></head><body>Измерение миграции и образования биопленки различных бактерий

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

Измерение миграции и образования биопленки различных бактерий

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their ...

Anti-Infective Efficacy in Cystic Fibrosis with Aggregate Biofilm Models

Cystic Fibrosis Aggregate Biofilm Model to Study Infection-relevant Gene Expression

Standard pre-clinical testing methods for novel antimicrobial therapeutics used to treat chronic lung infections in people with cystic fibrosis do not reflect the environmental conditions of the hostile lung niche. Current reductionist testing conditions can lead to the progression of compounds along a preclinical pipeline without evidence of their activity under cystic fibrosis lung niche-appropriate conditions. Several approaches used to study traditional antimicrobials may not be suitable for antibiotic alternatives, including anti-virulence therapeutics like anti-quorum sensing agents and siderophore inhibitors. This protocol documents an aggregate biofilm model of Pseudomonas aeruginosa to compare resistance and infection-relevant gene expression in single-species and multi-species cultures (Staphylococcus aureus and Candida albicans), examining colony-forming unit (CFU) reductions and changes in gene expression, using algD as an exemplar. The model was optimized for small, static volumes of bacterial cultures to allow the study of novel compounds in the discovery phase of the drug development pipeline, where compound quantities may be limited. Single-species P. aeruginosa biofilms were formed in Synthetic Cystic Fibrosis Medium 2 (SCFM2) for 24 h before treatment with meropenem at different concentrations (1, 16, and 256 &#181;g/mL) for a further 24 h. Polymicrobial biofilms were established by growing Staphylococcus aureus and Candida albicans together in SCFM2, then inoculating with P. aeruginosa for an additional 24 h and treating with meropenem. The lack of a direct connection between compound efficacy measures in pre-clinical testing and clinical trial results has cast doubt on the applicability of current laboratory screening tools. This model allows us to understand the impact of relevant factors on P. aeruginosa gene expression, including genes contributing to resistance and virulence, thereby bridging this gap.

This study outlines a framework for developing an aggregate polymicrobial biofilm model and optimizing an RNA extraction protocol to assess Pseudomonas aeruginosa gene expression reflective of the cystic fibrosis lung environment. Applications include evaluating antimicrobial effects and studying antibiotic alternatives under conditions relevant to cystic fibrosis.

Standard pre-clinical testing methods for novel antimicrobial therapeutics used to treat chronic lung infections in people with cystic fibrosis do not ...

Visualization of Internalized Anaerobic Bacteria within Host Endothelial Cells

Source: Wunsch, C. M. et. al., <a href="https://app.jove.com/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
In this video, we demonstrate a procedure to visualize internalized Porphyromonas gingivalis, an anaerobic pathogenic bacterium, within human endothelial cells using confocal microscopy. The bacteria are stained with a fluorescent dye while the host endothelial cell actin cytoskeleton is labeled with a different fluorescent dye. The internalized Porphyromonas gingivalis inside endothelial cells are visualized using confocal microscopy.

Porphyromonas gingivalis в качестве модельного организма для оценки интерактивности анаэробных бактерий с клеток-хозяев

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