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 biofilms7, 8. 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.
20 Related JoVE Articles!
Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)
Institutions: University of Ottawa.
This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro
. Bacteria are inoculated into the wells of a 96-well microtiter plate. In the case of the planktonic assay, serial dilutions of the antimicrobial agent of choice are added to the bacterial suspensions. In the biofilm assay, once inoculated, the bacteria are left to form a biofilm over a set period of time. Unattached cells are removed from the wells, the media is replenished and serial dilutions of the antimicrobial agent of choice are added. After exposure to the antimicrobial agent, the planktonic cells are assayed for growth. For the biofilm assay, the media is refreshed with fresh media lacking the antimicrobial agent and the biofilm cells are left to recover. Biofilm cell viability is assayed after the recovery period. The MBC-P for the antimicrobial agent is defined as the lowest concentration of drug that kills the cells in the planktonic culture. In contrast, the MBC-B for a strain is determined by exposing preformed biofilms to increasing concentrations of antimicrobial agent for 24 hr. The MBC-B is defined as the lowest concentration of antimicrobial agent that kills the cells in the biofilm.
Immunology, Issue 83, biofilm, planktonic, antibiotic resistance, static, antibacterial, minimal inhibitory concentration (MIC)
Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells
Institutions: Dartmouth College, Indiana University Purdue University Indianapolis.
Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa
biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa
is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa
and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa
strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa
infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa
biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.
Cellular Biology, Issue 44, biofilm, Pseudomonas aeruginosa, airway, epithelial cells, co-culture, cytotoxicity, Cystic Fibrosis, virulence
Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat
Institutions: Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Geffen School of Medicine at UCLA.
Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3
. Staphylococcus aureus
is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6
. The increase in infections due to methicillin-resistant S. aureus
(MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8
. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.
Infection, Issue 64, Immunology, Staphylococcus aureus, endocarditis, animal model, methicillin resistance, MRSA, rat
Microtiter Dish Biofilm Formation Assay
Institutions: Dartmouth Medical School.
Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae
, Escherichia coli
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa
. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.
Immunology, Issue 47, Biofilm, assay, bacteria, fungi, microtiter, static
Determining the Contribution of the Energy Systems During Exercise
Institutions: University of Sao Paulo, University of Sao Paulo, University of Sao Paulo, University of Sao Paulo.
One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1
(mL . s-1
) x t1
(s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete’s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.
Physiology, Issue 61, aerobic metabolism, anaerobic alactic metabolism, anaerobic lactic metabolism, exercise, athletes, mathematical model
Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model
Institutions: London School of Hygiene & Tropical Medicine.
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.
Infection, Issue 80, Gram-Negative Bacteria, Bacterial Infections, Gastrointestinal Diseases, Campylobacter jejuni, bacterial invasion, intestinal epithelial cells, models of infection
Culturing and Maintaining Clostridium difficile in an Anaerobic Environment
Institutions: Emory University School of Medicine.
is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile
is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile
from fecal samples and subsequently culturing C. difficile
for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile
growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile
Immunology, Issue 79, Genetics, Bacteria, Anaerobic, Gram-Positive Endospore-Forming Rods, Spores, Bacterial, Gram-Positive Bacterial Infections, Clostridium Infections, Bacteriology, Clostridium difficile, Gram-positive, anaerobic chamber, spore, culturing, maintenance, cell culture
The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins
Institutions: Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine.
Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e.
endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.
Basic Protocol, Issue 82, Endocytosis, recycling, plasma membrane, cell surface, EZLink, Sulfo-NHS-SS-Biotin, L-Glutathione, GSH, thiol group, disulfide bond, epithelial cells, cell polarization
Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species
Institutions: University of British Columbia .
The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia
species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia
species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.
Immunology, Issue 80, Bacteria, Aerobic, Gram-Negative Bacteria, Immune System Diseases, Respiratory Tract Diseases, Burkholderia, proteins, glass beads, 2-D gel electrophoresis
Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
Institutions: University of Virginia Health System.
Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2
. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella
species, and Escherichia coli3-5
, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8
. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants.
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9
. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10
, is ideal for LPS extraction.
Immunology, Issue 63, Microbiology, Gram-negative, LPS, extraction, polysaccharide staining, Western immunoblot
The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms
Institutions: Bar-Ilan University, Bar-Ilan University, Bar-Ilan University, Bar-Ilan University.
Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5
. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7
. There are also reports of biocidal effect of red and near infra red8
as well as green light9
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa
. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa
is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa
strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa
. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa
was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.
Microbiology, Issue 77, Infection, Infectious Diseases, Cellular Biology, Molecular Biology, Biophysics, Chemistry, Biomedical Engineering, Bacteria, Photodynamic therapy, Medical optics, Bacterial viability, Antimicrobial treatment, Laser, Gentamycin, antibiotics, reactive oxygen species, pathogens, microorganisms, cell culture
Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis
Institutions: Marshall University.
is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa
is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa
is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa
. To better understand alginate regulation, we describe a protocol using the mini-himar1
transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1
transposon (pFAC) from host E. coli
SM10/λpir into recipient P. aeruginosa
biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas
isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE
(PA4033) and kinB
(PA5484), in strain PAO1 with a wild-type mucA
encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, σ22
). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.
Immunology, Issue 85, Pseudomonas aeruginosa, alginate, mucoidy, mutagenesis, mini-himar1 mariner transposon, pFAC
Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice
Institutions: San Raffaele Scientific Institute, Italian Cystic Fibrosis Research Foundation.
A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa
agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo
. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa
and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa
clinical strains in the agar-beads in vitro
, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa
RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa
colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.
Infection, Issue 85, Opportunistic Infections, Respiratory Tract Infections, Inflammation, Lung Diseases, Cystic Fibrosis, Pseudomonas aeruginosa
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
Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria
Institutions: Auburn University , Keesler Air Force Base.
A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus
strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus
. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3
. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus
strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.
Bioengineering, Issue 75, Microbiology, Infectious Diseases, Infection, Medicine, Immunology, Cellular Biology, Molecular Biology, Genetics, Anatomy, Physiology, Bacteria, Pharmacology, Staphylococcus, Bacteriophages, phage, Binding, Competitive, Biophysics, surface properties (nonmetallic materials), surface wave acoustic devices (electronic design), sensors, Lytic phage spheroids, QCM-D, Langmuir-Blodgett (LB) monolayers, MRSA, Staphylococcus aureus, assay
Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing
Institutions: University Hospital Center and University of Lausanne.
Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.
Immunology, Issue 92, blood culture, bacteriology, identification, antibiotic susceptibility testing, MALDI-TOF MS.
Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
Institutions: Institut Pasteur .
RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3
. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7
Immunology, Issue 52, Polymerase fidelity, RNA virus, mutation frequency, mutagen, RNA polymerase, viral evolution
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
Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae
Institutions: University of Manchester.
Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to <40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae
. Like prokaryotic systems, S. cerevisiae
can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-β-D-maltoside (DDM) and 1-tetradecanoyl-sn
-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.
Biochemistry, Issue 87, Membrane protein, cystic fibrosis, CFTR, ABCC7, protein purification, Cystic Fibrosis Foundation, green fluorescent protein
Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes
Institutions: Johns Hopkins University.
In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.
Cellular Biology, Issue 5, Translational Research, mosquito, malaria, virus, dengue, genetics, injection, RNAi, transgenesis, transgenic