Antibiotic Sensitivity Testing
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Antibiotic sensitivity testing or antibiotic susceptibility testing is the measurement of the susceptibility of
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
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. It is used because bacteria may have resistance to some antibiotics. Sensitivity testing results can allow a clinician to change the choice of antibiotics from
empiric therapy Empiric therapy or empirical therapy is medical treatment or therapy based on experience and, more specifically, therapy begun on the basis of a clinical "educated guess" in the absence of complete or perfect information. Thus it is applied before ...
, which is when an antibiotic is selected based on clinical suspicion about the site of an infection and common causative bacteria, to
directed therapy Directed therapy refers to the treatment of infections based on specific knowledge of what the causal agent is able to be treated with. It is the opposite to empiric therapy, which refers to the treatment of infections based on the clinical suspicio ...
, in which the choice of antibiotic is based on knowledge of the organism and its sensitivities. Sensitivity testing usually occurs in a
medical laboratory A medical laboratory or clinical laboratory is a laboratory where tests are conducted out on clinical specimens to obtain information about the health of a patient to aid in diagnosis, treatment, and prevention of disease. Clinical Medical labor ...
, and uses
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 ...
methods that expose bacteria to antibiotics, or genetic methods that test to see if bacteria have genes that confer resistance. Culture methods often involve measuring the diameter of areas without bacterial growth, called zones of inhibition, around paper discs containing antibiotics on agar culture dishes that have been evenly inoculated with bacteria. The
minimum inhibitory concentration In microbiology, the minimum inhibitory concentration (MIC) is the lowest concentration of a chemical, usually a drug, which prevents visible growth of a bacterium or bacteria. MIC depends on the microorganism, the affected human being (in vivo onl ...
, which is the lowest concentration of the antibiotic that stops the growth of bacteria, can be estimated from the size of the zone of inhibition. Antibiotic susceptibility testing has been needed since the discovery of the
beta-lactam A beta-lactam (β-lactam) ring is a four-membered lactam. A ''lactam'' is a cyclic amide, and ''beta''-lactams are named so because the nitrogen atom is attached to the β-carbon atom relative to the carbonyl. The simplest β-lactam possible is ...
antibiotic
penicillin Penicillins (P, PCN or PEN) are a group of β-lactam antibiotics originally obtained from ''Penicillium'' moulds, principally '' P. chrysogenum'' and '' P. rubens''. Most penicillins in clinical use are synthesised by P. chrysogenum using ...
. Initial methods were phenotypic, and involved culture or dilution. The
Etest Etest (previously known as the Epsilometer test) is a way of determining antimicrobial sensitivity by placing a strip impregnated with antimicrobials onto an agar plate. A strain of bacterium or fungus will not grow near a concentration of anti ...
, an antibiotic impregnated strip, has been available since the 1980s, and genetic methods such as
polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
(PCR) testing have been available since the early 2000s. Research is ongoing into improving current methods by making them faster or more accurate, as well as developing new methods for testing, such as
microfluidics Microfluidics refers to the behavior, precise control, and manipulation of fluids that are geometrically constrained to a small scale (typically sub-millimeter) at which surface forces dominate volumetric forces. It is a multidisciplinary field tha ...
.


Uses

In clinical medicine, antibiotics are most frequently prescribed on the basis of a person's
symptom Signs and symptoms are the observed or detectable signs, and experienced symptoms of an illness, injury, or condition. A sign for example may be a higher or lower temperature than normal, raised or lowered blood pressure or an abnormality showin ...
s and
medical guideline Medicine is the science and practice of caring for a patient, managing the diagnosis, prognosis, prevention, treatment, palliation of their injury or disease, and promoting their health. Medicine encompasses a variety of health care practi ...
s. This method of antibiotic selection is called
empiric therapy Empiric therapy or empirical therapy is medical treatment or therapy based on experience and, more specifically, therapy begun on the basis of a clinical "educated guess" in the absence of complete or perfect information. Thus it is applied before ...
, and it is based on knowledge about what bacteria cause an infection, and to what antibiotics bacteria may be sensitive or resistant. For example, a simple
urinary tract infection A urinary tract infection (UTI) is an infection that affects part of the urinary tract. When it affects the lower urinary tract it is known as a bladder infection (cystitis) and when it affects the upper urinary tract it is known as a kidney ...
might be treated with
trimethoprim/sulfamethoxazole Trimethoprim/sulfamethoxazole, sold under the brand name Bactrim among others, is a fixed-dose combination antibiotic medication used to treat a variety of bacterial infections. It consists of one part trimethoprim to five parts sulfamethoxazo ...
. This is because ''
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'' is the most likely causative bacterium, and may be sensitive to that
combination antibiotic A combination antibiotic is one in which two ingredients are added together for additional therapeutic effect. One or both ingredients may be antibiotics. Antibiotic combinations are increasingly important because of antimicrobial resistance. This ...
. However, bacteria can be resistant to several classes of
antibiotics 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 o ...
. This resistance might be because a type of bacteria has intrinsic resistance to some antibiotics, because of resistance following past exposure to antibiotics, or because resistance may be transmitted from other sources such as
plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
s. Antibiotic sensitivity testing provides information about which antibiotics are more likely to be successful and should therefore be used to treat the infection. Antibiotic sensitivity testing is also conducted at a population level in some countries as a form of
screening Screening may refer to: * Screening cultures, a type a medical test that is done to find an infection * Screening (economics), a strategy of combating adverse selection (includes sorting resumes to select employees) * Screening (environmental), a ...
. This is to assess the background rates of resistance to antibiotics (for example with methicillin-resistant ''Staphylococcus aureus''), and may influence guidelines and
public health Public health is "the science and art of preventing disease, prolonging life and promoting health through the organized efforts and informed choices of society, organizations, public and private, communities and individuals". Analyzing the det ...
measures.


Methods

Once a bacterium has been identified following
microbiological culture A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture medium under controlled laboratory conditions. Microbial cultures are foundational and basic diagn ...
, antibiotics are selected for susceptibility testing. Susceptibility testing methods are based on exposing bacteria to antibiotics and observing the effect on the growth of the bacteria (phenotypic testing), or identifying specific genetic markers (genetic testing). Methods used may be qualitative, meaning that a result indicates resistance is or is not present; or quantitative, using a
minimum inhibitory concentration In microbiology, the minimum inhibitory concentration (MIC) is the lowest concentration of a chemical, usually a drug, which prevents visible growth of a bacterium or bacteria. MIC depends on the microorganism, the affected human being (in vivo onl ...
(MIC) to describe the concentration of antibiotic to which a bacterium is sensitive. There are many factors that can affect the results of antibiotic sensitivity testing, including failure of the instrument, temperature, moisture, and potency of the antimicrobial agent.
Quality control Quality control (QC) is a process by which entities review the quality of all factors involved in production. ISO 9000 defines quality control as "a part of quality management focused on fulfilling quality requirements". This approach places ...
(QC) testing helps to ensure the accuracy of test results. Organizations such as the
American Type Culture Collection ATCC or the American Type Culture Collection is a nonprofit organization which collects, stores, and distributes standard reference microorganisms, cell lines and other materials for research and development. Established in 1925 to serve as a natio ...
and
National Collection of Type Cultures National Collection of Type Cultures (NCTC) is one of the four culture collections that constitutes the Culture Collections operated by Public Health England. It is a non-profit culture collection repository located in the UK. NCTC maintains ove ...
provide strains of bacteria with known resistance phenotypes that can be used for quality control.


Phenotypic methods

Testing based on exposing bacteria to antibiotics uses
agar plate An agar plate is a Petri dish that contains a growth medium solidified with agar, used to culture microorganisms. Sometimes selective compounds are added to influence growth, such as antibiotics. Individual microorganisms placed on the plate wil ...
s or dilution in agar or broth. The selection of antibiotics will depend on the organism grown, and the antibiotics that are available locally. To ensure that the results are accurate, the concentration of bacteria that is added to the agar or broth (the
inoculum In biology, inoculum refers to the source material used for inoculation. ''Inoculum'' may refer to: * In medicine, material that is the source of the inoculation in a vaccine * In microbiology, propagules: cells, tissue, or viruses that are used t ...
) must be standardized. This is accomplished by comparing the
turbidity Turbidity is the cloudiness or haziness of a fluid caused by large numbers of individual particles that are generally invisible to the naked eye, similar to smoke in air. The measurement of turbidity is a key test of water quality. Fluids can ...
of bacteria suspended in saline or broth to
McFarland standards In microbiology, McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range to standardize microbial testing. An example of such testing is antibiotic s ...
—solutions whose turbidity is equivalent to that of a suspension containing a given concentration of bacteria. Once an appropriate concentration (most commonly an 0.5 McFarland standard) has been reached, which can be determined by visual inspection or by
photometry Photometry can refer to: * Photometry (optics), the science of measurement of visible light in terms of its perceived brightness to human vision * Photometry (astronomy), the measurement of the flux or intensity of an astronomical object's electrom ...
, the inoculum is added to the
growth medium A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss ''Physcomitrella patens''. Differen ...
.


Manual

The disc diffusion method involves selecting a strain of bacteria, placing it on an agar plate, and observing bacterial growth near antibiotic-impregnated discs. This is also called the Kirby-Bauer method, although modified methods are also used. In some cases,
urine sample A urine test is any medical test performed on a urine specimen. The analysis of urine is a valuable diagnostic tool because its composition reflects the functioning of many body systems, particularly the kidneys and urinary system, and specimens a ...
s or positive
blood culture A blood culture is a medical laboratory test used to detect bacteria or fungi in a person's blood. Under normal conditions, the blood does not contain microorganisms: their presence can indicate a bloodstream infection such as bacteremia or f ...
samples are applied directly to the test medium, bypassing the preliminary step of isolating the organism. If the antibiotic inhibits microbial growth, a clear ring, or zone of inhibition, is seen around the disc. The bacteria are classified as sensitive, intermediate, or resistant to an antibiotic by comparing the diameter of the zone of inhibition to defined thresholds which correlate with MICs.
Mueller–Hinton agar Mueller–Hinton agar is a microbiological growth medium that is commonly used for antibiotic susceptibility testing, specifically disk diffusion tests. It is also used to isolate and maintain ''Neisseria'' and ''Moraxella'' species. It typi ...
is frequently used in the disc diffusion test. The
Clinical and Laboratory Standards Institute The Clinical and Laboratory Standards Institute (CLSI) is a volunteer-driven, membership-supported, not-for-profit, standards development organization. CLSI promotes the development and use of voluntary laboratory consensus standards and guideline ...
(CLSI) and
European Committee on Antimicrobial Susceptibility Testing European Committee on Antimicrobial Susceptibility Testing (EUCAST) is a scientific committee for defining guidelines to interpret antimicrobial resistance. It was formed in 1997 and is jointly organized by ESCMID, ECDC and other European labora ...
(EUCAST) provide standards for the type and depth of agar, temperature of incubation, and method of analysing results. Disc diffusion is considered the cheapest and most simple of the methods used to test for susceptibility, and is easily adapted to testing newly available antibiotics or formulations. Some slow-growing and fastidious bacteria cannot be accurately tested by this method, while others, such as ''Streptococcus'' species and ''
Haemophilus influenzae ''Haemophilus influenzae'' (formerly called Pfeiffer's bacillus or ''Bacillus influenzae'') is a Gram-negative, non-motile, coccobacillary, facultatively anaerobic, capnophilic pathogenic bacterium of the family Pasteurellaceae. The bacteria ...
'', can be tested but require specialized growth media and incubation conditions. Gradient methods, such as
Etest Etest (previously known as the Epsilometer test) is a way of determining antimicrobial sensitivity by placing a strip impregnated with antimicrobials onto an agar plate. A strain of bacterium or fungus will not grow near a concentration of anti ...
, use a plastic strip placed on agar. A plastic strip impregnated with different concentrations of antibiotics is placed on a growth medium, and the growth medium is viewed after a period of incubation. The minimum inhibitory concentration can be identified based on the intersection of the teardrop-shaped zone of inhibition with the marking on the strip. Multiple strips for different antibiotics may be used. This type of test is considered a diffusion test. In agar and broth dilution methods, bacteria are placed in multiple small tubes with different concentrations of antibiotics. Whether a bacterium is sensitive or not is determined by visual inspection or automatic optical methods, after a period of incubation. Broth dilution is considered the gold standard for phenotypic testing. The lowest concentration of antibiotics that inhibits growth is considered the MIC.


Automated

Automated systems exist that replicate manual processes, for example, by using imaging and software analysis to report the zone of inhibition in diffusion testing, or dispensing samples and determining results in dilutional testing. Automated instruments, such as the VITEK 2, BD Phoenix, and Microscan systems, are the most common methodology for AST. The specifications of each instrument vary, but the basic principle involves the introduction of a bacterial suspension into pre-formulated panels of antibiotics. The panels are incubated and the inhibition of bacterial growth by the antibiotic is automatically measured using methodologies such as
turbidimetry Turbidimetry (the name being derived from ''turbidity'') is the process of measuring the loss of intensity of transmitted light due to the scattering effect of particles suspended in it. Light is passed through a filter creating a light of known wav ...
,
spectrophotometry Spectrophotometry is a branch of electromagnetic spectroscopy concerned with the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. Spectrophotometry uses photometers, known as spec ...
or
fluorescence 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 ...
detection. An
expert system In artificial intelligence, an expert system is a computer system emulating the decision-making ability of a human expert. Expert systems are designed to solve complex problems by reasoning through bodies of knowledge, represented mainly as if ...
correlates the MICs with susceptibility results, and the results are automatically transmitted into the
laboratory information system A laboratory information management system (LIMS), sometimes referred to as a laboratory information system (LIS) or laboratory management system (LMS), is a software-based solution with features that support a modern laboratory's operations. K ...
for validation and reporting. While such automated testing is less labour-intensive and more standardized than manual testing, its accuracy can be comparatively poor for certain organisms and antibiotics, so the disc diffusion test remains useful as a backup method.


Genetic methods

Genetic testing, such as via
polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
(PCR),
DNA microarray A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to ...
, DNA chips, and
loop-mediated isothermal amplification Loop-mediated isothermal amplification (LAMP) is a single-tube technique for the amplification of DNA and a low-cost alternative to detect certain diseases. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combines LAMP with ...
, may be used to detect whether bacteria possess genes which confer antibiotic resistance. An example is the use of PCR to detect the
mecA ''mecA'' is a gene found in bacterial cells which allows them to be resistant to antibiotics such as methicillin, penicillin and other penicillin-like antibiotics. The bacteria strain most commonly known to carry ''mecA'' is methicillin-resista ...
gene for
beta-lactam A beta-lactam (β-lactam) ring is a four-membered lactam. A ''lactam'' is a cyclic amide, and ''beta''-lactams are named so because the nitrogen atom is attached to the β-carbon atom relative to the carbonyl. The simplest β-lactam possible is ...
resistant ''
Staphylococcus aureus ''Staphylococcus aureus'' is a Gram-positive spherically shaped bacterium, a member of the Bacillota, and is a usual member of the microbiota of the body, frequently found in the upper respiratory tract and on the skin. It is often positive ...
''. Other examples include assays for testing
vancomycin Vancomycin is a glycopeptide antibiotic medication used to treat a number of bacterial infections. It is recommended intravenously as a treatment for complicated skin infections, bloodstream infections, endocarditis, bone and joint infections, ...
resistance genes vanA and vanB in '' Enteroccocus'' species, and antibiotic resistance in ''
Pseudomonas aeruginosa ''Pseudomonas aeruginosa'' is a common encapsulated, gram-negative, aerobic–facultatively anaerobic, rod-shaped bacterium that can cause disease in plants and animals, including humans. A species of considerable medical importance, ''P. aerugi ...
'', ''
Klebsiella pneumoniae ''Klebsiella pneumoniae'' is a Gram-negative, non-motile, encapsulated, lactose-fermenting, facultative anaerobic, rod-shaped bacterium. It appears as a mucoid lactose fermenter on MacConkey agar. Although found in the normal flora of the mouth ...
'' and ''Escherichia coli''. These tests have the benefit of being direct and rapid, as compared with observable methods, and have a high likelihood of detecting a finding when there is one to detect. However, whether resistance genes are detected does not always match the resistance profile seen with phenotypic method. The tests are also expensive and require specifically trained personnel. Polymerase chain reaction is a method of identifying genes related to antibiotic susceptibility. In the PCR process, a bacterium's DNA is denatured and the two strands of the double helix separate. Primers specific to a sought-after gene are added to a solution containing the DNA, and a
DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
is added alongside a mixture containing molecules that will be needed (for example,
nucleotide Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules wi ...
s and
ion An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conven ...
s). If the relevant gene is present, every time this process runs, the quantity of the target gene will be doubled. After this process, the presence of the genes is demonstrated through a variety of methods including
electrophoresis Electrophoresis, from Ancient Greek ἤλεκτρον (ḗlektron, "amber") and φόρησις (phórēsis, "the act of bearing"), is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric fie ...
,
southern blot A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detecti ...
ting, and other
DNA sequencing DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. Th ...
analysis methods. DNA microarrays and chips use the binding of
complementary DNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a spe ...
to a target gene or nucleic acid sequence. The benefit of this is that multiple genes can be assessed simultaneously.


MALDI-TOF

Matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) is another method of susceptibility testing. This is a form of
time-of-flight mass spectrometry Time-of-flight mass spectrometry (TOFMS) is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined by a time of flight measurement. Ions are accelerated by an electric field of known strength. This acceleration result ...
, in which the molecules of a bacterium are subject to matrix-assisted laser desorption. The ionised particles are then accelerated, and spectral peaks recorded, producing an expression profile, which is capable of differentiating specific bacterial strains after being compared to known profiles. This includes, in the context of antibiotic susceptibility testing, strains such as
beta-lactamase Beta-lactamases, (β-lactamases) are enzymes () produced by bacteria that provide multi-resistance to beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, monobactams and carbapenems (ertapenem), although carbapenems ...
producing ''E coli''. MALDI-TOF is rapid and automated. There are limitations to testing in this format however; results may not match the results of phenotypic testing, and acquisition and maintenance is expensive.


Reporting

The results of the testing are reported as a table, sometimes called an antibiogram. Bacteria are marked as sensitive, resistant, or having intermediate resistance to an antibiotic based on the minimum inhibitory concentration (MIC), which is the lowest concentration of the antibiotic that stops the growth of bacteria. The MIC is compared to standard threshold values (called "breakpoints") for a given bacterium and antibiotic. Breakpoints for the same organism and antibiotic may differ based on the site of infection: for example, the CLSI generally defines ''
Streptococcus pneumoniae ''Streptococcus pneumoniae'', or pneumococcus, is a Gram-positive, spherical bacteria, alpha-hemolytic (under aerobic conditions) or beta-hemolytic (under anaerobic conditions), aerotolerant anaerobic member of the genus Streptococcus. They are ...
'' as sensitive to
intravenous Intravenous therapy (abbreviated as IV therapy) is a medical technique that administers fluids, medications and nutrients directly into a person's vein. The intravenous route of administration is commonly used for rehydration or to provide nutrie ...
penicillin if MICs are ≤0.06 μg/ml, intermediate if MICs are 0.12 to 1 μg/ml, and resistant if MICs are ≥2 μg/ml, but for cases of
meningitis Meningitis is acute or chronic inflammation of the protective membranes covering the brain and spinal cord, collectively called the meninges. The most common symptoms are fever, headache, and neck stiffness. Other symptoms include confusion or ...
, the breakpoints are considerably lower. Sometimes, whether an antibiotic is marked as resistant is also based on bacterial characteristics that are associated with known methods of resistance such as the potential for
beta-lactamase Beta-lactamases, (β-lactamases) are enzymes () produced by bacteria that provide multi-resistance to beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, monobactams and carbapenems (ertapenem), although carbapenems ...
production. Specific patterns of drug resistance or
multidrug resistance Multiple drug resistance (MDR), multidrug resistance or multiresistance is antimicrobial resistance shown by a species of microorganism to at least one antimicrobial drug in three or more antimicrobial categories. Antimicrobial categories are c ...
may be noted, such as the presence of an extended-spectrum beta lactamase. Such information may be useful to the clinician, who can change the empiric treatment to a tailored treatment that is directed only at the causative bacterium.


Clinical practice

Ideal antibiotic therapy is based on determining the causal agent and its antibiotic sensitivity. Empiric treatment is often started before laboratory microbiological reports are available. This might be for common or relatively minor infections based on clinical guidelines (such as
community-acquired pneumonia Community-acquired pneumonia (CAP) refers to pneumonia (any of several lung diseases) contracted by a person outside of the healthcare system. In contrast, hospital-acquired pneumonia (HAP) is seen in patients who have recently visited a hospital ...
), or for serious infections, such as
sepsis Sepsis, formerly known as septicemia (septicaemia in British English) or blood poisoning, is a life-threatening condition that arises when the body's response to infection causes injury to its own tissues and organs. This initial stage is follo ...
or
bacterial meningitis Meningitis is acute or chronic inflammation of the protective membranes covering the brain and spinal cord, collectively called the meninges. The most common symptoms are fever, headache, and neck stiffness. Other symptoms include confusion or ...
, in which delayed treatment carries substantial risks. The effectiveness of individual antibiotics varies with the anatomical site of the infection, the ability of the antibiotic to reach the site of infection, and the ability of the bacteria to resist or inactivate the antibiotic. Specimens for antibiotic sensitivity testing are ideally collected before treatment is started. A sample may be taken from the site of a suspected infection; such as a
blood culture A blood culture is a medical laboratory test used to detect bacteria or fungi in a person's blood. Under normal conditions, the blood does not contain microorganisms: their presence can indicate a bloodstream infection such as bacteremia or f ...
sample when bacteria are suspected to be present in the bloodstream (
bacteraemia Bloodstream infections (BSIs), which include bacteremias when the infections are bacterial and fungemias when the infections are fungal, are infections present in the blood. Blood is normally a sterile environment, so the detection of micro ...
), a
sputum Sputum is mucus that is coughed up from the lower airways (the trachea and bronchi). In medicine, sputum samples are usually used for a naked eye examination, microbiological investigation of respiratory infections and cytological investigations ...
sample in the case of a
pneumonia Pneumonia is an inflammatory condition of the lung primarily affecting the small air sacs known as alveoli. Symptoms typically include some combination of productive or dry cough, chest pain, fever, and difficulty breathing. The severity ...
, or a
urine Urine is a liquid by-product of metabolism in humans and in many other animals. Urine flows from the kidneys through the ureters to the urinary bladder. Urination results in urine being excretion, excreted from the body through the urethra. Cel ...
sample in the case of a
urinary tract infection A urinary tract infection (UTI) is an infection that affects part of the urinary tract. When it affects the lower urinary tract it is known as a bladder infection (cystitis) and when it affects the upper urinary tract it is known as a kidney ...
. Sometimes multiple samples may be taken if the source of an infection is not clear. These samples are transferred to the microbiology laboratory where they are added to
culture media A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss ''Physcomitrella patens''. Different ...
, in or on which the bacteria grow until they are present in sufficient quantities for identification and sensitivity testing to be carried out. When antibiotic sensitivity testing is completed, it will report the organisms present in the sample, and which antibiotics they are susceptible to. Although antibiotic sensitivity testing is done in a laboratory (
in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...
), the information provided about this is often clinically relevant to the antibiotics in a person (
in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...
). Sometimes, a decision must be made for some bacteria as to whether they are the cause of an infection, or simply
commensal Commensalism is a long-term biological interaction (symbiosis) in which members of one species gain benefits while those of the other species neither benefit nor are harmed. This is in contrast with mutualism, in which both organisms benefit fro ...
bacteria or contaminants, such as ''
Staphylococcus epidermidis ''Staphylococcus epidermidis'' is a Gram-positive bacterium, and one of over 40 species belonging to the genus '' Staphylococcus''. It is part of the normal human microbiota, typically the skin microbiota, and less commonly the mucosal microbio ...
'' and other
opportunistic infection An opportunistic infection is an infection caused by pathogens (bacteria, fungi, parasites or viruses) that take advantage of an opportunity not normally available. These opportunities can stem from a variety of sources, such as a weakened immune ...
s. Other considerations may influence the choice of antibiotics, including the need to penetrate through to an infected site (such as an
abscess An abscess is a collection of pus that has built up within the tissue of the body. Signs and symptoms of abscesses include redness, pain, warmth, and swelling. The swelling may feel fluid-filled when pressed. The area of redness often extends b ...
), or the suspicion that one or more causes of an infection were not detected in a sample.


History

Since the discovery of the
beta-lactam A beta-lactam (β-lactam) ring is a four-membered lactam. A ''lactam'' is a cyclic amide, and ''beta''-lactams are named so because the nitrogen atom is attached to the β-carbon atom relative to the carbonyl. The simplest β-lactam possible is ...
antibiotic
penicillin Penicillins (P, PCN or PEN) are a group of β-lactam antibiotics originally obtained from ''Penicillium'' moulds, principally '' P. chrysogenum'' and '' P. rubens''. Most penicillins in clinical use are synthesised by P. chrysogenum using ...
, the rates of antimicrobial resistance have increased. Over time, methods for testing the sensitivity of bacteria to antibiotics have developed and changed.
Alexander Fleming Sir Alexander Fleming (6 August 1881 – 11 March 1955) was a Scottish physician and microbiologist, best known for discovering the world's first broadly effective antibiotic substance, which he named penicillin. His discovery in 1928 of w ...
in the 1920s developed the first method of susceptibility testing. The "gutter method" that he developed was a diffusion method, involving an antibiotic that was diffused through a gutter made of agar. In the 1940s, multiple investigators, including Pope, Foster and Woodruff, Vincent and Vincent used paper discs instead. All these methods involve testing only susceptibility to penicillin. The results were difficult to interpret and not reliable, because of inaccurate results that were not standardised between laboratories. Dilution has been used as a method to grow and identify bacteria since the 1870s, and as a method of testing the susceptibility of bacteria to antibiotics since 1929, also by Alexander Fleming. The way of determining susceptibility changed from how turbid the solution was, to the pH (in 1942), to optical instruments. The use of larger tube-based "macrodilution" testing has been superseded by smaller "microdilution" kits. In 1966, the
World Health Organisation The World Health Organization (WHO) is a specialized agency of the United Nations responsible for international public health. The WHO Constitution states its main objective as "the attainment by all peoples of the highest possible level of h ...
confirmed the Kirby-Bauer method as the standard method for susceptibility testing; it is simple, cost-effective and can test multiple antibiotics. The Etest was developed in 1980 by Bolmstrӧm and Eriksson, and MALDI-TOF developed in 2000s. An array of automated systems has been developed since and after the 1980s. PCR was the first genetic test available and first published as a method of detecting antibiotic susceptibility in 2001.


Further research

Point-of-care testing Point-of-care testing (POCT or bedside testing) is defined as medical diagnostic testing at or near the point of care—that is, at the time and place of patient care. This contrasts with the historical pattern in which testing was wholly or most ...
is being developed to speed up the time for testing, and to help practitioners avoid prescribing unnecessary antibiotics in the style of
precision medicine Precision, precise or precisely may refer to: Science, and technology, and mathematics Mathematics and computing (general) * Accuracy and precision, measurement deviation from true value and its scatter * Significant figures, the number of digit ...
. Traditional techniques typically take between 12 and 48 hours, although it can take up to five days. In contrast, rapid testing using
molecular diagnostics Molecular diagnostics is a collection of techniques used to analyze biological markers in the genome and proteome, and how their cells express their genes as proteins, applying molecular biology to medical testing. In medicine the technique is ...
is defined as "being feasible within an 8-h(our) working shift". Progress has been slow due to a range of reasons including cost and regulation. Additional research is focused at the shortcomings of current testing methods. As well as the duration it takes to report phenotypic methods, they are laborious, have difficult portability and are difficult to use in resource-limited settings, and have a chance of cross-contamination. As of 2017, point-of-care resistance diagnostics were available for
methicillin-resistant Staphylococcus aureus Methicillin-resistant ''Staphylococcus aureus'' (MRSA) is a group of Gram-positive bacteria that are genetically distinct from other strains of ''Staphylococcus aureus''. MRSA is responsible for several difficult-to-treat infections in humans. ...
(MRSA),
rifampin Rifampicin, also known as rifampin, is an ansamycin antibiotic used to treat several types of bacterial infections, including tuberculosis (TB), ''Mycobacterium avium'' complex, leprosy, and Legionnaires’ disease. It is almost always used tog ...
-resistant
Mycobacterium tuberculosis ''Mycobacterium tuberculosis'' (M. tb) is a species of pathogenic bacteria in the family Mycobacteriaceae and the causative agent of tuberculosis. First discovered in 1882 by Robert Koch, ''M. tuberculosis'' has an unusual, waxy coating on its c ...
(TB), and vancomycin-resistant enterococci (VRE) through GeneXpert by molecular diagnostics company
Cepheid A Cepheid variable () is a type of star that pulsates radially, varying in both diameter and temperature and producing changes in brightness with a well-defined stable period and amplitude. A strong direct relationship between a Cepheid varia ...
. Quantitative PCR, with the view of determining the percent of a detected bacteria that possesses a resistance gene, is being explored.
Whole genome sequencing Whole genome sequencing (WGS), also known as full genome sequencing, complete genome sequencing, or entire genome sequencing, is the process of determining the entirety, or nearly the entirety, of the DNA sequence of an organism's genome at a s ...
of isolated bacteria is also being explored, and likely to become more available as costs decrease and speed increases over time. Additional methods explored include
microfluidics Microfluidics refers to the behavior, precise control, and manipulation of fluids that are geometrically constrained to a small scale (typically sub-millimeter) at which surface forces dominate volumetric forces. It is a multidisciplinary field tha ...
, which uses a small amount of fluid and a variety of testing methods, such as optical, electrochemical, and magnetic. Such assays do not require much fluid to be tested, are rapid and portable. The use of fluorescent dyes has been explored. These involve labelled proteins targeted at
biomarker In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, ...
s, nucleic acid sequences present within cells that are found when the bacterium is resistant to an antibiotic. An isolate of bacteria is fixed in position and then dissolved. The isolate is then exposed to fluorescent dye, which will be luminescent when viewed. Improvements to existing platforms are also being explored, including improvements in imaging systems that are able to more rapidly identify the MIC in phenotypic samples; or the use of bioluminescent enzymes that reveal bacterial growth to make changes more easily visible.


Bibliography

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References


External links

* {{Clinical microbiology techniques Antibiotics Microbiology techniques Infectious diseases