The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.
22 Related JoVE Articles!
Using Coculture to Detect Chemically Mediated Interspecies Interactions
Institutions: University of North Carolina at Chapel Hill .
In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e.
biofilm formation, sporulation, virulence factor production, etc
.) Screening is performed under growth conditions where this phenotype is not
expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis
; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.
Microbiology, Issue 80, High-Throughput Screening Assays, Genes, Reporter, Microbial Interactions, Soil Microbiology, Coculture, microbial interactions, screen, fluorescent transcriptional reporters, Bacillus subtilis
Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection
Institutions: Imperial College London.
, the causative agent of a severe pneumonia named Legionnaires' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella
containing vacuole (LCV). L. pneumophila
infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella
are suitable for investigation of L. pneumophila
infection. G. mellonella
is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae's immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila
is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila
virulence in the G. mellonella
model, including how to grow infectious L. pneumophila
, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila
virulence, describing a new tool to aid our understanding of this complex pathogen.
Infection, Issue 81, Bacterial Infections, Infection, Disease Models, Animal, Bacterial Infections and Mycoses, Galleria mellonella, Legionella pneumophila, insect model, bacterial infection, Legionnaires' disease, haemocytes
Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays
Institutions: BioSentinel Inc., Madison, WI.
Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay's use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro
replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT's proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.
Neuroscience, Issue 85, Botulinum, food testing, detection, quantification, complex matrices, BoTest Matrix, Clostridium, potency testing
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example
Institutions: University of Houston, University of Arkansas.
Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes
. With respect to L. monocytogenes
, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e.
deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g.
small farms), hospitals, nursing homes, cruise ships, and hotels.
Environmental Sciences, Issue 85, cross contamination, retail deli, fluorescent powder, Listeria monocytogenes, foodborne pathogens
Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water
Institutions: University of Wyoming, Colorado State University, Colorado State University, Colorado State University, University of California, Davis, University of Florida, McGill University.
This protocol describes rapid colorimetric detection of Escherichia coli
spp., and Listeria monocytogenes
from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (µPADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the µPAD. Alternatively, digital images of the reacted µPADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24 hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.
Environmental Sciences, Issue 88, Paper-based analytical device (µPAD), Colorimetric enzymatic detection, Salmonella spp., Listeria monocytogenes, Escherichia coli, Modified Moore Swab (MMS), agricultural water, food safety, environmental microbiology
A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites
Institutions: Boston University School of Medicine, Boston University School of Medicine.
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.
Immunology, Issue 90,
Pathogen-Induced Chronic Inflammation; Porphyromonas gingivalis; Oral Bone Loss; Periodontal Disease; Atherosclerosis; Chronic Inflammation; Host-Pathogen Interaction; microCT; MRI
Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions
Institutions: Imperial College London, Institut Pasteur, Unité Macrophages et Développement de l'Immunité.
is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri
by compartmentalizing bacteria inside ‘septin cages’ and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro
using tissue culture cells and in vivo
using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.
Infection, Issue 91, ATG8/LC3, autophagy, cytoskeleton, HeLa cells, p62, septin, Shigella, zebrafish
Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection
Institutions: Center for Research and Advanced Studies of the National Polytechnic Institute, Center for Research and Advanced Studies of the National Polytechnic Institute, Center for Research and Advanced Studies of the National Polytechnic Institute.
is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica
invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica
invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.
Immunology, Issue 88, Entamoeba histolytica, EhCPADH112, cell adhesion, MDCK, Caco-2, tight junction disruption, amoebiasis, host-pathogen interaction, infection model, actin cytoskeleton
In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration
Institutions: Harvard Medical School, MGH for Children, Massachusetts General Hospital.
Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state. Cocultured in vitro
models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro
model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa
(PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli
(MC1000). The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro
model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro
coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.
Infection, Issue 83, Cellular Biology, Epithelium, Neutrophils, Pseudomonas aeruginosa, Respiratory Tract Diseases, Neutrophils, epithelial barriers, pathogens, transmigration
Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection
Institutions: University of Maine, University of Maine.
Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo
, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans
, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans
. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.
Immunology, Issue 93, Zebrafish, mucosal candidiasis, mucosal infection, epithelial barrier, epithelial cells, innate immunity, swimbladder, Candida albicans, in vivo.
Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food
Institutions: University of Kentucky .
are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes
. Using this model, mice fed L. monocytogenes
-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes
to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.
Infection, Issue 75, Microbiology, Immunology, Infectious Diseases, Genetics, Cellular Biology, Medicine, Biomedical Engineering, Anatomy, Physiology, Pathology, Surgery, Listeria, animal models, Bacteria, intestines, food borne pathogen, L. monocytogenes, bacterial pathogens, inoculation, isolation, cell culture, mice, animal model
Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants
Institutions: Boyce Thompson Institute for Plant Research, Cornell University.
To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize conserved microbial features called pathogen-associated molecular patterns (PAMPs) and this detection leads to PAMP-triggered immunity (PTI), which effectively prevents colonization of plant tissues by non-pathogens1,2
. The most well studied system in PTI is the FLS2
. FLS2 recognizes the PAMP flg22 that is a component of bacterial flagellin.
Successful pathogens possess virulence factors or effectors that can suppress PTI and allow the pathogen to cause disease1
. Some plants in turn possess resistance genes that detect effectors or their activity, which leads to effector-triggered immunity (ETI)2
We describe a cell death-based assay for PTI modified from Oh and Collmer4
. The assay was standardized in N. benthamiana
, which is being used increasingly as a model system for the study of plant-pathogen interactions5
. PTI is induced by infiltration of a non-pathogenic bacterial strain into leaves. Seven hours later, a bacterial strain that either causes disease or which activates ETI is infiltrated into an area overlapping the original infiltration zone. PTI induced by the first infiltration is able to delay or prevent the appearance of cell death due to the second challenge infiltration. Conversely, the appearance of cell death in the overlapping area of inoculation indicates a breakdown of PTI.
Four different combinations of inducers of PTI and challenge inoculations were standardized (Table 1). The assay was tested on non-silenced N. benthamiana
plants that served as the control and plants silenced for FLS2
that were predicted to be compromised in their ability to develop PTI.
Jove Infectious Diseases, Plant Biology, Issue 31, plant immunity, pathogen-associated molecular pattern (PAMP), PAMP-triggered immunity (PTI), effector-triggered immunity (ETI), Nicotiana benthamiana
Invasion of Human Cells by a Bacterial Pathogen
Institutions: University of Bath.
Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus
. The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus
would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2
at 37°C. For S. aureus
this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 μl tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.
Infection, Issue 49, Bacterial pathogen, host cell invasion, Staphylococcus aureus, invasin
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung
Institutions: University of Liverpool , University of Liverpool .
There is growing concern about the relevance of in vitro
antimicrobial susceptibility tests when applied to isolates of P. aeruginosa
from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1
. However, during chronic lung infections in CF, P. aeruginosa
populations exist in biofilms and there is evidence that the environment is largely microaerophilic2
. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3
Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa
growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6
. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa
based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
An ASM assay was developed in a microtitre plate format. P. aeruginosa
biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa
isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a >128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3
. Several in vitro
models have been used previously to study P. aeruginosa
. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9
. In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2
and affect antibiotic susceptibility10
. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.
Immunology, Issue 64, Microbiology, Pseudomonas aeruginosa, antimicrobial susceptibility, artificial sputum media, lung infection, cystic fibrosis, diagnostics, plankton
Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count
Institutions: McGill University, Montreal, QC Canada.
Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. QDs have attracted a lot of interest for imaging, diagnostics, and therapy due to their bright, stable fluorescence1,2 3,4,5
. QDs can be conjugated to a variety of bio-active molecules for binding to bacteria and mammalian cells6
QDs are also being widely investigated as cytotoxic agents for targeted killing of bacteria. The emergence of multiply-resistant bacterial strains is rapidly becoming a public health crisis, particularly in the case of Gram negative pathogens 7
. Because of the well-known antimicrobial effect of certain nanomaterials, especially Ag, there are hundreds of studies examining the toxicity of nanoparticles to bacteria 8
. Bacterial studies have been performed with other types of semiconductor nanoparticles as well, especially TiO2 9,10-11
, but also ZnO12
and others including CuO 13
. Some comparisons of bacterial strains have been performed in these studies, usually comparing a Gram negative strain with a Gram positive. With all of these particles, mechanisms of toxicity are attributed to oxidation: either the photogeneration of reactive oxygen species (ROS) by the particles or the direct release of metal ions that can cause oxidative toxicity. Even with these materials, results of different studies vary greatly. In some studies the Gram positive test strain is reportedly more sensitive than the Gram negative 10
; in others it is the opposite 14
. These studies have been well reviewed 15
In all nanoparticle studies, particle composition, size, surface chemistry, sample aging/breakdown, and wavelength, power, and duration of light exposure can all dramatically affect the results. In addition, synthesis byproducts and solvents must be considered16 17
. High-throughput screening techniques are needed to be able to develop effective new nanomedicine agents.
CdTe QDs have anti-microbial effects alone18
or in combination with antibiotics. In a previous study, we showed that coupling of antibiotics to CdTe can increase toxicity to bacteria but decrease toxicity to mammalian cells, due to decreased production of reactive oxygen species from the conjugates19
. Although it is unlikely that cadmium-containing compounds will be approved for use in humans, such preparations could be used for disinfection of surfaces or sterilization of water.
In this protocol, we give a straightforward approach to solubilizing CdTe QDs with mercaptopropionic acid (MPA). The QDs are ready to use within an hour. We then demonstrate coupling to an antimicrobial agent.
The second part of the protocol demonstrates a 96-well bacterial inhibition assay using the conjugated and unconjugated QDs. The optical density is read over many hours, permitting the effects of QD addition and light exposure to be evaluated immediately as well as after a recovery period. We also illustrate a colony count for quantifying bacterial survival.
Biomedical Engineering, Issue 65, Bioengineering, Molecular Biology, Quantum dots, solubilization, conjugation, cytotoxicity, phototoxicity, growth curve, plate count
The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis
Institutions: INRA, Micalis UMR1319, France.
The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1
. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3
. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1
. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella
have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum
, Aspergillus fumigatus
, Candida albicans
) and bacterial pathogens, such as Staphylococcus aureus
, Proteus vulgaris
, Serratia marcescens Pseudomonas aeruginosa
, Listeria monocytogenes
or Enterococcus faecalis4-7
. Regardless of the bacterial species, results obtained with Galleria
larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria
, and strains causing severe human infections are also highly virulent in the Galleria
. Oral infection of Galleria
is much less used and additional compounds, like specific toxins, are needed to reach mortality.
larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 °C to 30 °C) and infection studies can be conducted between 15 °C to above 37 °C12,13
, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014
, measurement of bacterial survival15,16
and examination of the infection process17
. Here, we describe the rearing of the insects, covering all life stages of G. mellonella
. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus
, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19
Infection, Issue 70, Microbiology, Immunology, Molecular Biology, Bacteriology, Entomology, Bacteria, Galleria mellonella, greater wax moth, insect larvae, intra haemocoelic injection, ingestion, animal model, host pathogen interactions
Microgavage of Zebrafish Larvae
Institutions: University of North Carolina at Chapel Hill .
The zebrafish has emerged as a powerful model organism for studying intestinal development1-5
, and host-microbe interactions17-25
. Experimental approaches for studying intestinal biology often require the in vivo
introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26
. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results.
We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g.
genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.
Biochemistry, Issue 72, Molecular Biology, Anatomy, Physiology, Basic Protocols, Surgery, Zebrafish, Danio rerio, intestine, lumen, larvae, gavage, microgavage, epithelium, barrier function, gut motility, microsurgery, microscopy, animal model
Live-cell Video Microscopy of Fungal Pathogen Phagocytosis
Institutions: University of Aberdeen, University of Aberdeen.
Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5
but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8
; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7
; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.
Infection, Issue 71, Immunology, Microbiology, Medicine, Cellular Biology, Molecular Biology, Infectious Diseases, Mycoses, Candidiasis, Bacterial Infections and Mycoses, Immune System Diseases, Live-cell imaging, phagocytosis, Candida albicans, host-pathogen interaction, pathogen, pathogen-associated molecular patterns, pattern recognition receptors, macrophage, fungus
Vibrio cholerae: Model Organism to Study Bacterial Pathogenesis - Interview
Institutions: University of California Santa Cruz - UCSC.
Microbiology, issue 4, microbial community, Vibrio cholerae, genome
Studies of Bacterial Chemotaxis Using Microfluidics - Interview
Institutions: MIT - Massachusetts Institute of Technology.
Microbiology, issue 4, microbial community, chemotaxis, microfluidics