O financiamento para este trabalho foi fornecida pelas Ciências Naturais e Engenharia Council of Canada (NSERC) eo Sentinela Rede papel bioativo.

<ol>
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A maioria dos métodos de detecção de bactérias comuns hoje são ou lenta (microbiana clássico) ou tecnicamente exigentes (anticorpo, PCR). Assim, acreditamos que a próxima geração de ferramentas de detecção deve atender para velocidade e simplicidade. Para este fim, criámos um RNA-clivagem e fluorescência de sinalização DNAzyme que pode ser usado para desenvolver ensaios simples para relatar a presença de bactérias através da geração de um sinal de fluorescência. O destaque DNAzyme sonda, RFD-EC1, é ativado pela CEM produzido durante o crescimento de E. coli em meios de cultura. Uma vez que o nosso método utiliza bruto misturas extracelulares de uma bactéria como o alvo de detecção e ignora a extracção alvo trabalhoso e passos de amplificação, ele pode ser usado para definir-se muito simples, &quot;mistura-e-ler&quot; tipo de ensaios para a detecção de bactérias. A utilização da nossa DNAzyme não se restringe a método de detecção de fluorescência com base. Por exemplo, a detecção colorimétrica utilizando o mesmo sistema de ensaio c DNAzymeum ser concebido usando um método relatado anteriormente que explora a amplificação círculo rolante em conjunção com um pigmento orgânico. 32 Nós prever a utilização de DNAzymes para a detecção de bactérias como uma avenida atraente para gerar novos biossensores bacterianas com maior simplicidade operacional.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo ou modelo </td></tr><tr><td> Agar </td><td> BioShop Canadá </td><td> AGR003 </td></tr><tr><td> Persulfato de amônio (APS) </td><td> BioShop Canadá </td><td> AMP001 </td></tr><tr><td> Acrilamida / bis-acrilamida (40%, 29:1) </td><td> BioShop Canadá </td><td> ACR004 </td></tr><tr><td> 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> 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> EMDChemicals </td><td> B10149-34 </td></tr><tr><td> NaCl </td><td> BioShop Canadá </td><td> SOD002 </td></tr><tr><td> NaOAc </td><td> Merck Química </td><td> SXO255-1 </td></tr><tr><td> NaOH </td><td> Merck Química </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> Uréia </td><td> BioShop Canadá </td><td> URE001 </td></tr><tr><td> Xylenecyanol FF </td><td> Sigma-Aldrich </td><td> X4126 </td></tr><tr><td> Concentrador de DNA </td><td> Thermo Scientific </td><td> Savant DNA Speedvac 120 </td></tr><tr><td> Millex unidade de filtro </td><td> Millipore </td><td> SLGP033RS </td></tr><tr><td> Dicas para a colocação de gel </td&gt; <td> DiaMed </td><td> TEC200EX-K </td></tr><tr><td> Software ImageQuant </td><td> Dinâmica Molecular </td><td> Versão 5.0 </td></tr><tr><td> Kimwipes </td><td> Kimberly-Clark Professional </td><td> 34705 </td></tr><tr><td> Mini Vortexer </td><td> VWR </td><td> 58816-121 </td></tr><tr><td> Parafilm </td><td> Pechiney Embalagens Plásticas </td><td> PM996 </td></tr><tr><td> As placas de Petri </td><td> Fisher Scientific </td><td> Fisherbrand 08-757-12 </td></tr><tr><td> Stripettor Plus (Pipetar arma) </td><td> Corning </td><td> 07764714 </td></tr><tr><td> Cubetas de quartzo </td><td> Varian Inc </td><td> 66-100216-00 </td></tr><tr><td> Shaker / Incubadora </td><td> New Brunswick Scientific </td><td> Série Classic C24 </td></tr><tr><td> Typhoon Scanner </td><td> GE Healthcare </td><td> 9200 modo variável </td></tr><tr><td> Centrifugar </td><td> Beckman Coulter </td><td> Allegra X22-R </td></tr><tr><td> Espectrofotômetro UV </td><td> Thermo Scientific </td><td> GenesysUV 10 </td></tr><tr><td> Espectrofotômetro de Fluorescência </td><td> Varian Inc </td><td> Cary Eclipse </td></tr></tbody></table>

detection of bacteria using fluorogenic dnazymes

Surtos ligados à transmitidas por alimentos e hospitalar e para conta de patógenos por milhões de mortes e internações, bem como colossais perdas econômicas a cada ano. Prevenção da ocorrência de tais surtos e minimização do impacto de um lugar epidemia em curso uma demanda cada vez maior de métodos analíticos que podem identificar com precisão patógenos culpado na primeira fase. Embora exista uma grande variedade de métodos eficazes para a detecção de patógenos, nenhum deles pode satisfazer todos os seguintes cinco requisitos principais incorporados por um método de detecção ideal: alta especificidade (detectando apenas a bactéria de interesse), de alta sensibilidade (capaz de detectar como baixa como uma célula viva único bacteriana), curto tempo-para-resultados (de minutos a horas), grande simplicidade operacional (sem necessidade de procedimentos de amostragem longos eo uso de equipamentos especializados), e rentabilidade. Por exemplo, de métodos microbiológicos clássicos são altamente específico, mas requer um tempo longo (days para semanas) para adquirir um resultado definitivo. 1 PCR e anticorpo de técnicas baseadas em oferecer os tempos de espera mais curtos (horas a dias), mas requerem a utilização de reagentes dispendiosos e / ou equipamento sofisticado. 2-4 Consequentemente, existe ainda uma grande demanda para a pesquisa científica para o desenvolvimento de inovadores métodos de detecção de bactérias que oferecem características melhoradas em um ou mais dos requisitos acima referidos. Nosso laboratório está interessado em examinar o potencial de DNAzymes como uma nova classe de sondas moleculares para aplicações biosensoriamento incluindo a detecção de bactérias 5. DNAzymes (também conhecido como deoxyribozymes ou enzimas de ADN) são feitas pelo homem moléculas de cadeia simples de ADN com a capacidade de catalisar reacções químicas. 6-8 Estas moléculas podem ser isoladas a partir de uma piscina de DNA vasta sequência aleatória (que contém até 10 16 seqüências individuais) por um processo conhecido como &quot;seleção in vitro&quot; or &quot;SELEX&quot; (evolução sistemática de ligantes por enriquecimento exponencial). 9-16 Estas moléculas de DNA especiais têm sido amplamente analisados ​​nos últimos anos como ferramentas moleculares para aplicações biosensoriamento 6-8. O nosso laboratório estabeleceu em procedimentos de selecção in vitro para isolar RNA-clivagem DNAzymes fluorescentes (RFDs;. Fig. 1) e investigou a utilização de RFDs como ferramentas analíticas 17-29 RFDs catalisar a clivagem de um substrato de ADN RNA-quimérico em um ribonucleótido único. junção (R) que é flanqueada por um fluoróforo (F) e um supressor (Q). A proximidade estreita do F e Q processa a fluorescência do substrato não clivado mínima. No entanto, o evento de clivagem conduz à separação de F e Q, o qual é acompanhado por um aumento significativo da intensidade de fluorescência. Mais recentemente, foi desenvolvido um método de isolar RFDs para a detecção de bactérias. 5 Estes RFDs especiais foram isolados para &quot;acender &quot;, na presença da mistura em bruto extracelular (CEM) deixado para trás por um tipo específico de bactérias no seu ambiente ou nos meios de comunicação que são cultivadas (Fig. 1). A utilização de mistura bruta contorna o tedioso processo de purificação e a identificação de um alvo adequado a partir do micróbio de interesse para o desenvolvimento de biossensores (que poderia levar meses ou anos para completar). A utilização de alvos extracelulares, o procedimento ensaio é simples porque não há necessidade de passos para obter alvos intracelulares. Usando o método acima, nós derivado uma RFD que cliva o seu substrato (FS1;. Fig. 2A) apenas na presença do CEM produzido por E. coli (CEM-CE). 5 Este E. coli com detecção RFD, chamado RFD-EC1 (Fig. 2A), foi encontrada a ser estritamente responsivo a CEM-CE, mas não responsiva ao CEM a partir de um hospedeiro de outras bactérias (Fig. 3). Aqui nós Present os procedimentos fundamentais experimentais para criação de E. coli ensaios de detecção utilizando resultados de RFD-EC1 e representativa.

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

Detection of Bacteria Using Fluorogenic DNAzymes

Recentemente, relatou uma nova abordagem para a geração de sondas DNAzyme fluorogênicos que podem ser aplicadas para criar um simples, &quot;mistura-e-ler&quot; teste de fluorescência para a detecção bacteriana. Estas sondas de DNA especiais catalisar a clivagem de um cromóforo-modificado substrato DNA-RNA quimérico, na presença de mistura extracelular em bruto (CEM) produzida por uma bactéria específica, assim, traduzindo a detecção de bactérias em geração do sinal de fluorescência. Neste relatório, irá descrever-chave procedimentos experimentais onde uma sonda específica DNAzyme denotado &quot;RFD-EC1&quot; é utilizado para a detecção da bactéria modelo,<em&gt; Escherichia coli (E. coli)</em&gt;.

Detecção de bactérias utilizando DNAzymes fluorogénicas

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

18.9K visualizações.  McMaster University. Recentemente, relatou uma nova abordagem para a geração de sondas DNAzyme fluorogênicos que podem ser aplicadas para criar um simples, "mistura-e-ler" teste de fluorescência para a detecção bacteriana. Estas sondas de DNA especiais catalisar a clivagem de um cromóforo-modificado substrato DNA-RNA quimérico, na presença de mistura extracelular em bruto (CEM) produzida por uma bactéria específica, assim, traduzindo a detecção de bactérias em geraçã...

Vídeo: 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).

Combinação de adesivos de fita baseada em amostragem e de fluorescência In situ Hibridação para detecção rápida de 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 ...

Imunologia e Infecção

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.

Detecção de multiplex de bactérias em amostras clínicas e Complexo Ambiental utilizando Oligonucleotídeos-coupled 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.

Rápida identificação de bactérias gram-negativas de Sangue Cultura Caldo Usando MALDI-TOF Espectrometria de Massa

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.

A detecção in situ de bactérias dentro dos tecidos de parafina-encaixados Usando um marcado com Digoxina DNA Probe Segmentação 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.

Detecção ultra-sensível de biomarcadores usando um Imprinting Molecular baseado Biosensor capacitivo

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.

Uma plataforma de alto rendimento para o rastreio 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 ...

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

Análise para inorgânico polifosfato em bactérias

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

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.

Quantificando partículas semelhantes a vírus norovírus humanos ligando-se a bactérias commensal usando citometria de fluxo

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 colonização vaginal murina por bactérias cultivadas anaerobicamente

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

Rastreamento de patógenos de órgãos inteiros por meio de quantificação fluorescente de repórter

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 em destaque: Desvendando as respostas bacterianas aos antibióticos e ao sistema imunológico nos tecidos

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

Tomografia tridimensional de órgãos inteiros de alta resolução de infecções microbianas

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