La financiación de este trabajo fue proporcionado por las Ciencias Naturales e Ingeniería de Investigación de Canadá (NSERC) y la Red de Médicos Centinelas de papel bioactivo.

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No hay conflictos de interés declarado.

La mayoría de los métodos de detección de bacterias comunes hoy en día son o bien lento (microbiana clásica) o técnicamente exigente (anticuerpos, PCR). Por lo tanto, creemos que la próxima generación de herramientas de detección debe atender a la velocidad y la simplicidad. Para este fin, se ha creado una ARN-escisión y la fluorescencia de señalización DNAzyme que puede utilizarse para desarrollar ensayos simples para informar la presencia de bacterias a través de la generación de una señal de fluorescencia. La sonda equipado DNAzyme, RFD-EC1, es activado por el CEM producidos durante el crecimiento de E. coli en medios de cultivo. Puesto que nuestro método utiliza crudo mezclas extracelulares de una bacteria como el objetivo de detección y evita la extracción de destino laborioso y pasos de amplificación, que puede ser utilizado para configurar muy simple &quot;, mezclar y leer&quot; tipo de ensayos para la detección bacteriana. El uso de nuestro DNAzyme no se limita al método de detección basado en fluorescencia. Por ejemplo, la detección colorimétrica utilizando el mismo sistema de ensayo DNAzyme cun ser diseñado usando un método ya se ha informado que explota amplificación círculo rodante en conjunción con un colorante orgánico. 32 Se prevé el uso de ADNzimas para la detección bacteriana como una atractiva vía para generar nuevos biosensores bacterianos con mayor simplicidad operativa.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo o modelo </td></tr><tr><td> Agar </td><td> Bioshop Canadá </td><td> AGR003 </td></tr><tr><td> Persulfato de amonio (APS) </td><td> Bioshop Canadá </td><td> AMP001 </td></tr><tr><td> La acrilamida / bis-acrilamida (40%, 29:1) </td><td> Bioshop Canadá </td><td> ACR004 </td></tr><tr><td> El ácido bórico </td><td> Bioshop Canadá </td><td> BOR001 </td></tr><tr><td> Azul de bromofenol </td><td> Sigma-Aldrich </td><td> B8026 </td></tr><tr><td> EDTA </td><td> EM Science </td><td> EXO539-1 </td></tr><tr><td> De HCl </td><td> Sigma-Aldrich </td><td> 38281 </td></tr><tr><td> HEPES </td><td> Bioshop Canadá </td><td> HEP001 </td></tr><tr><td> Caldo LB </td><td> Sigma-Aldrich </td><td> L3022 </td></tr><tr><td> MgCl 2 </td><td> EMDProductos Químicos </td><td> B10149-34 </td></tr><tr><td> NaCl </td><td> Bioshop Canadá </td><td> SOD002 </td></tr><tr><td> NaOAc </td><td> Merck Chemicals </td><td> SXO255-1 </td></tr><tr><td> NaOH </td><td> Merck Chemicals </td><td> SXO590-1 </td></tr><tr><td> SDS </td><td> Bioshop Canadá </td><td> SDS001 </td></tr><tr><td> TEMED </td><td> Bioshop Canadá </td><td> TEM001 </td></tr><tr><td> Tris-base </td><td> Bioshop Canadá </td><td> BST666 </td></tr><tr><td> Tween 20 </td><td> Sigma-Aldrich </td><td> P9416 </td></tr><tr><td> Urea </td><td> Bioshop Canadá </td><td> URE001 </td></tr><tr><td> Xylenecyanol FF </td><td> Sigma-Aldrich </td><td> X4126 </td></tr><tr><td> ADN concentrador </td><td> Thermo Scientific </td><td> Savant ADN SpeedVac 120 </td></tr><tr><td> Millex filtro de la unidad </td><td> Millipore </td><td> SLGP033RS </td></tr><tr><td> Gel puntas de carga </td&gt; <td> DiaMed </td><td> TEC200EX-K </td></tr><tr><td> ImageQuant software </td><td> Dinámica Molecular </td><td> La versión 5.0 </td></tr><tr><td> Kimwipes </td><td> Kimberly-Clark Professional </td><td> 34705 </td></tr><tr><td> Mini vórtice </td><td> VWR </td><td> 58816-121 </td></tr><tr><td> Parafilm </td><td> Pechiney Plastic Packaging </td><td> PM996 </td></tr><tr><td> Placas de Petri </td><td> Fisher Scientific </td><td> Fisherbrand 08-757-12 </td></tr><tr><td> Stripettor Plus (con una pipeta arma de fuego) </td><td> Corning </td><td> 07764714 </td></tr><tr><td> Cubetas de cuarzo </td><td> Varian Inc. </td><td> 66-100216-00 </td></tr><tr><td> Shaker / Incubadora </td><td> New Brunswick Scientific </td><td> Classic Series C24 </td></tr><tr><td> El tifón escáner </td><td> GE Healthcare </td><td> 9200 modo Variable </td></tr><tr><td> Centrifugar </td><td> Beckman Coulter </td><td> Allegra X22-R </td></tr><tr><td> Espectrofotómetro UV </td><td> Thermo Scientific </td><td> GenesysUV 10 </td></tr><tr><td> Espectrofotómetro de fluorescencia </td><td> Varian Inc. </td><td> Cary Eclipse </td></tr></tbody></table>

detection of bacteria using fluorogenic dnazymes

Los brotes vinculados a transmitidas por los alimentos y la adquirida en el hospital cuenta de los agentes patógenos de millones de muertes y hospitalizaciones, así como colosales pérdidas económicas cada año. La prevención de estos brotes y la minimización del impacto de la epidemia de un lugar permanente a una demanda cada vez mayor de métodos analíticos que permitan identificar con precisión los agentes patógenos causantes en la primera etapa. Aunque hay una gran variedad de métodos eficaces para la detección de patógenos, ninguno de ellos puede satisfacer todos los siguientes cinco requisitos principales enunciados de un método de detección ideal: alta especificidad (detección de la bacteria sólo de intereses), de alta sensibilidad (capaz de detectar un precio tan bajo como una sola célula bacteriana viva), corto tiempo de los resultados (de minutos a horas), una gran sencillez de funcionamiento (sin necesidad de largos procedimientos de muestreo y el uso de equipo especializado), y la rentabilidad. Por ejemplo, los métodos clásicos microbiológicos son altamente específicos, sino que requieren un tiempo largo (days a semana) para adquirir un resultado definitivo. 1 PCR y técnicas basadas en anticuerpos ofrecen tiempos de espera (horas a días), pero requieren el uso de reactivos caros y / o equipo sofisticado. 2-4 En consecuencia, existe todavía una gran demanda para la investigación científica hacia el desarrollo de innovadores métodos de detección de bacterias que ofrecen características mejoradas en uno o más de los requisitos mencionados anteriormente. Nuestro laboratorio está interesado en examinar el potencial de ADNzimas como una nueva clase de sondas moleculares para aplicaciones de biosensores, incluyendo la detección de bacterias. 5 ADNzimas (también conocido como deoxyribozymes o enzimas de ADN) son hechos por el hombre de cadena sencilla moléculas de ADN con la capacidad de catalizar reacciones químicas. 6-8 Estas moléculas se pueden aislar de una piscina de ADN al azar vasta-secuencia (que contiene hasta 10 16 secuencias individuales) mediante un proceso conocido como &quot;selección in vitro&quot; Or &quot;SELEX&quot; (evolución sistemática de ligandos por enriquecimiento exponencial). 9-16 Estas moléculas de ADN especiales han sido ampliamente examinados en los últimos años como herramientas moleculares para aplicaciones biosensores. 6-8 Nuestro laboratorio ha establecido en los procedimientos de selección in vitro para el aislamiento de ARN-escisión ADNzimas fluorescentes (DSR,. Fig. 1) e investigó el uso de la DSR como herramientas analíticas 17-29 DSR catalizan la escisión de un sustrato de ADN-ARN quimérico en una sola ribonucleótido. cruce (R) que está flanqueado por un fluoróforo (F) y un extintor (Q). La proximidad de F y Q representa la fluorescencia mínima sustrato no escindido. Sin embargo, el evento de escisión conduce a la separación de F y Q, que está acompañado por un incremento significativo de la intensidad de fluorescencia. Más recientemente, hemos desarrollado un método para aislar DSR para la detección de bacterias. 5 Estos DSR especiales fueron aislados a &quot;iluminar &quot;en la presencia de la mezcla bruta extracelular (CEM), dejada atrás por un tipo específico de bacterias en su medio ambiente o en los medios que se cultivan (fig. 1). El uso de la mezcla bruta evita el tedioso proceso de purificación y la identificación de un objetivo adecuado del microbio de interés para el desarrollo de biosensores (que podría tomar meses o años para completar). El uso de objetivos extracelulares, el procedimiento es simple ensayo, porque no hay necesidad de adoptar medidas para obtener los objetivos intracelulares. Utilizando el método anterior, se deriva una RFD que escinde su sustrato (FS1;. Fig. 2A) sólo en presencia del CEM producida por E. E. (CEM-CE). 5 E. Este E. detección RFD, llamado RFD-EC1 (Fig. 2A), se encontró que era estrictamente sensible a CEM-CE pero no responde a la CEM de una multitud de otras bacterias (fig. 3). Aquí se present los procedimientos experimentales clave para la creación de E. Los ensayos de detección de E. coli utilizando los resultados de RFD-EC1 y representativo.

Watch this Scientific Journal Video about Detection of Bacteria Using Fluorogenic DNAzymes at JoVE.com

Detection of Bacteria Using Fluorogenic DNAzymes

Recientemente hemos informado de un nuevo enfoque para la generación de sondas fluorogénicos DNAzyme que se pueden aplicar para crear un simple, &quot;mezclar y leer&quot; ensayo de inmunofluorescencia para la detección de bacterias. Estas sondas de ADN especiales catalizar la escisión de un cromóforo modificado con ADN-ARN sustrato quimérico en la presencia de la mezcla bruta extracelular (CEM), producida por una bacteria específica, con lo que la traducción de detección de bacterias en la generación de señales de fluorescencia. En este informe se describen los principales procedimientos experimentales que se emplea una sonda DNAzyme específica denominada &quot;RFD-EC1&quot; para la detección de la bacteria modelo,<em&gt; Escherichia coli (E. coli)</em&gt;.

La detección de bacterias mediante ADNzimas fluorogénicos

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

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Immunology and Infection

18.8K vistas.  McMaster University. Recientemente hemos informado de un nuevo enfoque para la generación de sondas fluorogénicos DNAzyme que se pueden aplicar para crear un simple, "mezclar y leer" ensayo de inmunofluorescencia para la detección de bacterias. Estas sondas de ADN especiales catalizar la escisión de un cromóforo modificado con ADN-ARN sustrato quimérico en la presencia de la mezcla bruta extracelular (CEM), producida por una bacteria específica, con lo que la traducción de d...

Video: Detection of Bacteria Using Fluorogenic DNAzymes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinación de adhesivo basado en cinta de muestreo y de fluorescencia In situ La hibridación para la detección rápida de Salmonella En los productos frescos

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Investigación

Inmunología e Infección

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Multiplex detección de bacterias en el complejo de muestras clínicas y ambientales utilizando oligonucleótidos de acoplamiento de microesferas fluorescentes

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

La identificación rápida de bacterias gram negativas de Cultura sangre caldo de cultivo utilizando MALDI-TOF espectrometría de masas

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

La detección in situ de las bacterias dentro de los tejidos embebidos en parafina utilizando un ADN sonda digoxina marcada focalización 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

Detección ultrasensible de biomarcadores utilizando una impresión Molecular basado Biosensor capacitiva

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Una plataforma de alto rendimiento para la detección de Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

Cuantificación de partículas similares al virus del norovirus humanos que se unen a bacterias comensales mediante citometría de flujo

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

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.

Modelos de colonización vaginal murina por bacterias cultivadas anaeróbicamente

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

Seguimiento de patógenos de todo el órgano mediante cuantificación de reporteros fluorescentes

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

Autor destacado: Desentrañando las respuestas bacterianas a los antibióticos y el sistema inmunitario en los tejidos

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

Tomografía tridimensional de órgano entero de alta resolución de infecciones microbianas

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...