The gentamicin protection assay or survival assay or invasion assay is a method used in

microbiology Microbiology () is the scientific study of microorganisms, those being unicellular (single cell), multicellular (cell colony), or acellular (lacking cells). Microbiology encompasses numerous sub-disciplines including virology, bacteriology, prot ...

. It is used to quantify the ability of

pathogen In biology, a pathogen ( el, πάθος, "suffering", "passion" and , "producer of") in the oldest and broadest sense, is any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a germ ...

bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...

to invade

eukaryotic cells Eukaryotes () are organisms whose Cell (biology), cells have a cell nucleus, nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the ...

. The assay is based on several observations made in the 1970s, in which the ability of internalized bacteria to avoid killing by

antibiotic An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention of ...

s was reported. The assay started to be used in

biological research Biology is the scientific study of life. It is a natural science with a broad scope but has several unifying themes that tie it together as a single, coherent field. For instance, all organisms are made up of cells that process hereditary in ...

in the early 1980s.

Background and principle

Intracellular bacteria need to enter

host cell In biology and medicine, a host is a larger organism that harbours a smaller organism; whether a parasitic, a mutualistic, or a commensalist ''guest'' (symbiont). The guest is typically provided with nourishment and shelter. Examples include a ...

s (

cells Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...

of the infected

organism In biology, an organism () is any living system that functions as an individual entity. All organisms are composed of cells (cell theory). Organisms are classified by taxonomy into groups such as multicellular animals, plants, and ...

) in order to replicate and propagate infection. Many species of ''

Shigella ''Shigella'' is a genus of bacteria that is Gram-negative, facultative anaerobic, non-spore-forming, nonmotile, rod-shaped, and genetically closely related to ''E. coli''. The genus is named after Kiyoshi Shiga, who first discovered it in 1897. ...

'' (causes

bacillary dysentery Bacillary dysentery is a type of dysentery, and is a severe form of shigellosis. It is associated with species of bacteria from the family Enterobacteriaceae. The term is usually restricted to ''Shigella'' infections. Shigellosis is caused by one ...

), ''

Salmonella ''Salmonella'' is a genus of rod-shaped (bacillus) Gram-negative bacteria of the family Enterobacteriaceae. The two species of ''Salmonella'' are ''Salmonella enterica'' and ''Salmonella bongori''. ''S. enterica'' is the type species and is fur ...

'' (

typhoid fever Typhoid fever, also known as typhoid, is a disease caused by '' Salmonella'' serotype Typhi bacteria. Symptoms vary from mild to severe, and usually begin six to 30 days after exposure. Often there is a gradual onset of a high fever over several ...

), ''

Mycobacterium ''Mycobacterium'' is a genus of over 190 species in the phylum Actinomycetota, assigned its own family, Mycobacteriaceae. This genus includes pathogens known to cause serious diseases in mammals, including tuberculosis ('' M. tuberculosis'') and ...

'' (

leprosy Leprosy, also known as Hansen's disease (HD), is a long-term infection by the bacteria ''Mycobacterium leprae'' or ''Mycobacterium lepromatosis''. Infection can lead to damage of the nerves, respiratory tract, skin, and eyes. This nerve damag ...

and

tuberculosis Tuberculosis (TB) is an infectious disease usually caused by '' Mycobacterium tuberculosis'' (MTB) bacteria. Tuberculosis generally affects the lungs, but it can also affect other parts of the body. Most infections show no symptoms, in ...

) and ''

Listeria ''Listeria'' is a genus of bacteria that acts as an intracellular parasite in mammals. Until 1992, 17 species were known, each containing two subspecies. By 2020, 21 species had been identified. The genus is named in honour of the British pi ...

'' (

listeriosis Listeriosis is a bacterial infection most commonly caused by '' Listeria monocytogenes'', although '' L. ivanovii'' and '' L. grayi'' have been reported in certain cases. Listeriosis can cause severe illness, including severe sepsis, men ...

), to name but a few, are intracellular. 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 Culture () is an umbrella term which encompasses the social behavior, institutions, and norms found in human societies, as well as the knowledge, beliefs, arts, laws, customs, capabilities, and habits of the individuals in these groups.Tyl ...

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 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 ...

gentamicin Gentamicin 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 e ...

Procedure

HeLa HeLa (; also Hela or hela) is an immortalized cell line used in scientific research. It is the oldest and most commonly used human cell line. The line is derived from cervical cancer cells taken on February 8, 1951, named after Henrietta La ...

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. The assay is performed in plastic

microtiter plates A microplate, also known as a microtiter plate (''Microtiter'' is a registered trademark in the United States, therefore it should not be used generically without attribution), microwell plate or multiwell, is a flat plate with multiple "wells" ...

, which are commonly used in

laboratories A laboratory (; ; colloquially lab) is a facility that provides controlled conditions in which scientific or technological research, experiments, and measurement may be performed. Laboratory services are provided in a variety of settings: physicia ...

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. 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 A detergent is a surfactant or a mixture of surfactants with cleansing properties when in dilute solutions. There are a large variety of detergents, a common family being the alkylbenzene sulfonates, which are soap-like compounds that are more ...

, most commonly

Triton X-100 Triton X-100 (''n'') is a nonionic surfactant that has a hydrophilic polyethylene oxide chain (on average it has 9.5 ethylene oxide units) and an aromatic hydrocarbon lipophilic or hydrophobic group. The hydrocarbon group is a 4-( 1,1,3,3-tetramet ...

. 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

gene In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...

s or

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

s to the bacteria's ability to invade cells can be easily assayed using this method. The gene in question can be

knocked out A knockout (abbreviated to KO or K.O.) is a fight-ending, winning criterion in several full-contact combat sports, such as boxing, kickboxing, muay thai, mixed martial arts, karate, some forms of taekwondo and other sports involving striking, a ...

, and the bacteria's invasiveness compared with that of normal,

wild type The wild type (WT) is the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "m ...

bacteria. Environmental conditions, such as pH level and

temperature Temperature is a physical quantity that expresses quantitatively the perceptions of hotness and coldness. Temperature is measured with a thermometer. Thermometers are calibrated in various temperature scales that historically have relied o ...

, 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: * Gentamicin can sometimes penetrate eukaryotic cells and kill the internalized bacteria. This may happen if the permeability of the cells somehow increased during the assay, sometimes due to poor handling of the cells. * Internalized bacteria may sometimes not be entirely protected from the outside environment, such as when the

phagosome In cell biology, a phagosome is a vesicle formed around a particle engulfed by a phagocyte via phagocytosis. Professional phagocytes include macrophages, neutrophils, and dendritic cells (DCs). A phagosome is formed by the fusion of the cell mem ...

(the

vacuole A vacuole () is a membrane-bound organelle which is present in plant and fungal cells and some protist, animal, and bacterial cells. Vacuoles are essentially enclosed compartments which are filled with water containing inorganic and organic mo ...

surrounding the bacterium inside the cell) is defective in some way. Gentamicin may kill those bacteria. * Gentamicin may fail to kill all the bacteria that remained outside the cells. 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 In biochemistry, immunostaining is any use of an antibody-based method to detect a specific protein in a sample. The term "immunostaining" was originally used to refer to the immunohistochemical staining of tissue sections, as first described by A ...

assay, based on the binding of

antibodies An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...

to bacteria before and after invasion. The antibodies emit

fluorescent Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...

, colored

light Light or visible light is electromagnetic radiation that can be perceived by the human eye. Visible light is usually defined as having wavelengths in the range of 400–700 nanometres (nm), corresponding to frequencies of 750–420 tera ...

, and the results of this assay are viewed under the

microscope A microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisibl ...

References

{{DEFAULTSORT:Gentamicin Protection Assay Microbiology techniques

Background and principle

Procedure

Usage, advantages and caveats

See also

References