Gentamicin protection assay

Last updated

The gentamicin protection assay or survival assay or invasion assay is a method used in microbiology. It is used to quantify the ability of pathogenic bacteria to invade eukaryotic cells.

Microbiology study of microscopic organisms

Microbiology is the study of microorganisms, those being unicellular, multicellular, or acellular. Microbiology encompasses numerous sub-disciplines including virology, parasitology, mycology and bacteriology.

In biology, a pathogen, in the oldest and broadest sense, is anything that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a germ.

Bacteria A domain of prokaryotes – single celled organisms without a nucleus

Bacteria are a type of biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a number of shapes, ranging from spheres to rods and spirals. Bacteria were among the first life forms to appear on Earth, and are present in most of its habitats. Bacteria inhabit soil, water, acidic hot springs, radioactive waste, and the deep portions of Earth's crust. Bacteria also live in symbiotic and parasitic relationships with plants and animals. Most bacteria have not been characterised, and only about half of the bacterial phyla have species that can be grown in the laboratory. The study of bacteria is known as bacteriology, a branch of microbiology.

Contents

The assay is based on several observations made in the 1970s, in which the ability of internalized bacteria to avoid killing by antibiotics was reported. [1] [2] The assay started to be used in biological research in the early 1980s.

Background and principle

Intracellular bacteria need to enter host cells (cells of the infected organism) in order to replicate and propagate infection. Many species of Shigella (causes bacillary dysentery), Salmonella (typhoid fever), Mycobacterium (leprosy and tuberculosis) and Listeria (listeriosis), to name but a few, are intracellular.

Cell (biology) the basic structural and functional unit of all organisms. Includes the plasma membrane and any external encapsulating structures such as the cell wall and cell envelope.

The cell is the basic structural, functional, and biological unit of all known living organisms. A cell is the smallest unit of life. Cells are often called the "building blocks of life". The study of cells is called cell biology or cellular biology.

Infection invasion of a host by disease-causing organisms

Infection is the invasion of an organism's body tissues by disease-causing agents, their multiplication, and the reaction of host tissues to the infectious agents and the toxins they produce. Infectious disease, also known as transmissible disease or communicable disease, is illness resulting from an infection.

Organism Any individual living physical entity

In biology, an organism is any system that exhibits the properties of life. It is a synonym for "life form".

Several antibiotics cannot penetrate eukaryotic cells. Therefore, these antibiotics cannot hurt intracellular bacteria that are already internalized. Using such antibiotics enables us to differentiate between bacteria that succeed in penetrating eukaryotic cells and those that do not. Applying such an antibiotic to a culture of eukaryotic cells infected with bacteria would kill the bacteria that remain outside the cells while sparing the ones that penetrated. The antibiotic of choice for this assay is the aminoglycoside gentamicin.

Cell culture process by which cells are grown under controlled conditions

Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature). Most cells require a surface or an artificial substrate (adherent or monolayer culture) whereas others can be grown free floating in culture medium (suspension culture). The lifespan of most cells is genetically determined, but some cell culturing cells have been “transformed” into immortal cells which will reproduce indefinitely if the optimal conditions are provided.

Aminoglycoside molecule or a portion of a molecule composed of amino-modified sugars

Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside (sugar). The term can also refer more generally to any organic molecule that contains amino sugar substructures. Aminoglycoside antibiotics display bactericidal activity against Gram-negative aerobes and some anaerobic bacilli where resistance has not yet arisen but generally not against Gram-positive and anaerobic Gram-negative bacteria.

Gentamicin chemical compound

Gentamicin, sold under brand name Garamycin among others, is an antibiotic used to treat several types of bacterial infections. This may include bone infections, endocarditis, pelvic inflammatory disease, meningitis, pneumonia, urinary tract infections, and sepsis among others. It is not effective for gonorrhea or chlamydia infections. It can be given intravenously, by injection into a muscle, or topically. Topical formulations may be used in burns or for infections of the outside of the eye. In the developed world, it is often only used for two days until bacterial cultures determine what specific antibiotics the infection is sensitive to. The dose required should be monitored by blood testing.

Procedure

The chemical structure of gentamicin Gentamicin C2.svg
The chemical structure of gentamicin

HeLa cells are commonly used as eukaryotic cells in the gentamicin protection assay, but other cells can be used as well. As for bacteria, only species susceptible to gentamicin can be assayed.

HeLa cell line

HeLa is an immortal cell line used in scientific research. It is the oldest and most commonly used human cell line. The line was derived from cervical cancer cells taken on February 8, 1951 from Henrietta Lacks, a patient who died of cancer on October 4, 1951. The cell line was found to be remarkably durable and prolific which warrants its extensive use in scientific research.

The assay is performed in plastic microtiter plates, which are commonly used in laboratories for culturing eukaryotic cells. The cells are allowed to grow in the wells overnight, creating a flat layer. Bacteria are separately grown overnight. On the next day the eukaryotic cells are inoculated with the bacteria and are incubated together for an hour. Centrifuging the plates for a few minutes may help bring cells and bacteria in contact and initiate infection.

Laboratory facility that provides controlled conditions in which scientific or technological research, experiments, and measurement may be performed.

A laboratory is a facility that provides controlled conditions in which scientific or technological research, experiments, and measurement may be performed.

The terms inoculation, vaccination, and immunization are often used synonymously to refer to artificial induction of immunity against various infectious diseases. However, there are some important historical and current differences. In English medicine, inoculation referred only to the practice of variolation until the very early 1800s. When Edward Jenner introduced smallpox vaccine in 1798, this was initially called cowpox inoculation or vaccine inoculation. Soon, to avoid confusion, smallpox inoculation continued to be referred to as variolation and cowpox inoculation was referred to as vaccination. Then, in 1891, Louis Pasteur proposed that the terms vaccine and vaccination should be extended to include the new protective procedures being developed. Immunization refers to the use of all vaccines but also extends to the use of antitoxin, which contains preformed antibody such as to diphtheria or tetanus exotoxins. Inoculation is now more or less synonymous in nontechnical usage with injection and the like, and questions along the lines of "Have you had your flu injection/vaccination/inoculation/immunization?" should not cause confusion. The focus is on what is being given and why, not the literal meaning of the technique used.

Centrifuge piece of equipment that puts an object in rotation around a fixed axis

A centrifuge is a piece of equipment that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis of spin (outward) that can be very strong. The centrifuge works using the sedimentation principle, where the centrifugal acceleration causes denser substances and particles to move outward in the radial direction. At the same time, objects that are less dense are displaced and move to the center. In a laboratory centrifuge that uses sample tubes, the radial acceleration causes denser particles to settle to the bottom of the tube, while low-density substances rise to the top.

After infection gentamicin is added to the plates, and they are incubated for an hour, allowing the antibiotic to kill all bacteria that were not able to penetrate the cells and remained outside. The plates are then washed well to remove the dead bacteria. Next the eukaryotic cells are lysed using a detergent, most commonly Triton X-100.

The bacteria that penetrated the cells and remained alive are now released, and they are plated on solid medium plates. Counting the colonies formed on the plates on the next day, and knowing how many bacteria were used in the beginning of the assay, enables the researcher to calculate the percentage of bacteria that were able to invade the eukaryotic cells.

Usage, advantages and caveats

The gentamicin protection assay is commonly used in pathogen research. The contribution of specific genes or proteins to the bacteria's ability to invade cells can be easily assayed using this method. The gene in question can be knocked out, and the bacteria's invasiveness compared with that of normal, wild type bacteria. Environmental conditions, such as pH level and temperature, can also be assayed for their effect on invasiveness.

The gentamicin protection assay is very sensitive, as it can detect the internalization of even single bacteria. It has several drawbacks:

To help assess the accuracy of a particular assay, positive and negative controls should be performed. When performing the assay as described above, bacteria that are known to be entirely invasive (positive control) and bacteria that are known as non-invasive (negative control) should be included in the assay.

An alternative invasion assay is the differential immunostaining assay, based on the binding of antibodies to bacteria before and after invasion. The antibodies emit fluorescent, colored light, and the results of this assay are viewed under the microscope.

See also

Related Research Articles

Tularemia primary bacterial infectious disease that has material basis in Francisella tularensis, which is transmitted by dog tick bite (Dermacentor variabilis), transmitted by deer flies (Chrysops sp) or transmitted by contact with infected animal tissues.

Tularemia, also known as rabbit fever, is an infectious disease caused by the bacterium Francisella tularensis. Symptoms may include fever, skin ulcers, and enlarged lymph nodes. Occasionally, a form that results in pneumonia or a throat infection may occur.

Lymphocyte Subtype of white blood cell

A lymphocyte is one of the subtypes of a white blood cell in a vertebrate's immune system. Lymphocytes include natural killer cells, T cells, and B cells. They are the main type of cell found in lymph, which prompted the name "lymphocyte".

<i>Chlamydia trachomatis</i> species of bacterium

Chlamydia trachomatis, commonly known as chlamydia, is a bacterium that can replicate only in human cells. It causes chlamydia, which can manifest in various ways, including: trachoma, lymphogranuloma venereum, nongonococcal urethritis, cervicitis, salpingitis, pelvic inflammatory disease. C. trachomatis is the most common infectious cause of blindness and the most common sexually transmitted bacterium.

<i>Listeria</i> genus of bacteria

Listeria is a genus of bacteria that, until 1992, contained 10 known species, each containing two subspecies. As of 2019, 20 species were identified. Named after the British pioneer of sterile surgery Joseph Lister, the genus received its current name in 1940. Listeria species are Gram-positive, rod-shaped, and facultatively anaerobic, and do not produce endospores. The major human pathogen in the genus Listeria is L. monocytogenes. It is usually the causative agent of the relatively rare bacterial disease listeriosis, an infection caused by eating food contaminated with the bacteria. Listeriosis can cause serious illness in pregnant women, newborns, adults with weakened immune systems and the elderly, and may cause gastroenteritis in others who have been severely infected.

<i>Legionella pneumophila</i> species of bacterium

Legionella pneumophila is a thin, aerobic, pleomorphic, flagellated, non-spore-forming, Gram-negative bacterium of the genus Legionella. L. pneumophila is the primary human pathogenic bacterium in this group and is the causative agent of Legionnaires' disease, also known as legionellosis.

Hemagglutination assay

The hemagglutination assay and the hemagglutination inhibition assay were developed in 1941–42 by American virologist George Hirst as methods for quantifying the relative concentration of viruses, bacteria, or antibodies.

Coccidia subclass of protists

Coccidia (Coccidiasina) are a subclass of microscopic, spore-forming, single-celled obligate intracellular parasites belonging to the apicomplexan class Conoidasida. As obligate intracellular parasites, they must live and reproduce within an animal cell. Coccidian parasites infect the intestinal tracts of animals, and are the largest group of apicomplexan protozoa.

Virulence factors are molecules produced by bacteria, viruses, fungi, and protozoa that add to their effectiveness and enable them to achieve the following:

<i>Anaplasma phagocytophilum</i> species of bacterium

Anaplasma phagocytophilum is a Gram-negative bacterium that is unusual in its tropism to neutrophils. It causes anaplasmosis in sheep and cattle, also known as tick-borne fever and pasture fever, and also causes the zoonotic disease human granulocytic anaplasmosis.

Medical microbiology medical specialty

Medical microbiology , the large subset of microbiology that is applied to medicine, is a branch of medical science concerned with the prevention, diagnosis and treatment of infectious diseases. In addition, this field of science studies various clinical applications of microbes for the improvement of health. There are four kinds of microorganisms that cause infectious disease: bacteria, fungi, parasites and viruses, and one type of infectious protein called prion.

G418 chemical compound

G418 (Geneticin) is an aminoglycoside antibiotic similar in structure to gentamicin B1. It is produced by Micromonospora rhodorangea. G418 blocks polypeptide synthesis by inhibiting the elongation step in both prokaryotic and eukaryotic cells. Resistance to G418 is conferred by the neo gene from Tn5 encoding an aminoglycoside 3'-phosphotransferase, APT 3' II. G418 is an analog of neomycin sulfate, and has similar mechanism as neomycin. G418 is commonly used in laboratory research to select genetically engineered cells. In general for bacteria and algae concentrations of 5 μg/mL or less are used, for mammalian cells concentrations of approximately 400 μg/mL are used for selection and 200 μg/mL for maintenance. However, optimal concentration for resistant clones selection in mammalian cells depends on the cell line used as well as on the plasmid carrying the resistance gene, therefore antibiotic titration should be done to find the best condition for every experimental system. Titration should be done using antibiotic concentrations ranging from 100 μg/mL up to 1400 μg/mL. Resistant clones selection could require from 1 to up to 3 weeks.

Bartonella bacilliformis is a proteobacterium, Gram negative aerobic, pleomorphic, flagellated, motile, coccobacillary, 2–3 μm long, 0.2–0.5 μm wide, and a facultative intracellular bacterium.

A pneumococcal infection is an infection caused by the bacterium Streptococcus pneumoniae, which is also called the pneumococcus. S. pneumoniae is a common member of the bacterial flora colonizing the nose and throat of 5–10% of healthy adults and 20–40% of healthy children. However, it is also a cause of significant disease, being a leading cause of pneumonia, bacterial meningitis, and sepsis. The World Health Organization estimate that in 2005 pneumococcal infections were responsible for the death of 1.6 million children worldwide.

Multidrug tolerance or antibiotic tolerance is the ability of a disease-causing microorganism to resist being killed by antibiotics or other antimicrobials. It is mechanistically distinct from multidrug resistance: It is not caused by mutant microbes, but rather by microbial cells that exist in a transient, dormant, non-dividing state. Microorganisms that display multidrug tolerance can be bacteria, fungi or parasites.

Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration. It is utilized in both research and development (R&D) in commercial and academic laboratories as well as production situations where the quantity of virus at various steps is an important variable. For example, the production of viral vaccines, recombinant proteins using viral vectors and viral antigens all require virus quantification to continually adapt and monitor the process in order to optimize production yields and respond to ever changing demands and applications. Examples of specific instances where known viruses need to be quantified include clone screening, multiplicity of infection (MOI) optimization and adaptation of methods to cell culture. This page discusses various techniques currently used to quantify viruses in liquid samples. These methods are separated into two categories, traditional vs. modern methods. Traditional methods are industry-standard methods that have been used for decades but are generally slow and labor-intensive. Modern methods are relatively new commercially available products and kits that greatly reduce quantification time. This is not meant to be an exhaustive review of all potential methods, but rather a representative cross-section of traditional methods and new, commercially available methods. While other published methods may exist for virus quantification, non-commercial methods are not discussed here.

The antibodies from lymphocyte secretions (ALS) assay is an immunological assay to detect active diseases like tuberculosis, cholera, typhoid etc. Recently, ALS assay nods the scientific community as it is rapidly used for diagnosis of Tuberculosis. The principle is based on the secretion of antibody from in vivo activated plasma B cells found in blood circulation for a short period of time in response to TB-antigens during active TB infection rather than latent TB infection.

References

  1. Mandell GL (1973). "Interaction of intraleukocytic bacteria and antibiotics". J Clin Invest. 52 (7): 1673–1679. doi:10.1172/JCI107348. PMC   302442 Lock-green.svg. PMID   4718959.
  2. Vaudaux P and Waldvogel FA (1979). "Gentamicin antibacterial activity in the presence of human polymorphonuclear leukocytes". Antimicrob Agents Chemother. 16 (6): 743–749. doi:10.1128/aac.16.6.743. PMC   352946 Lock-green.svg. PMID   533256.