A primary thesis of hedonism is that "pleasure is the good",
which leads to the argument that any component of life that is not
pleasurable does nothing directly to increase one's well-being. This is a view held by many value theorists, but most famously by some classical utilitarians. Nozick attacks the thesis by means of a thought experiment.
If he can show that there is something other than pleasure that has
value and thereby increases well-being, then hedonism is refuted.
The thought experiment
Nozick
describes a machine that could provide whatever desirable or
pleasurable experiences a subject could want. In this thought
experiment, psychologists have figured out a way to stimulate a person's
brain to induce pleasurable experiences that the subject could not
distinguish from those they would have apart from the machine. He then
asks, if given the choice, would the subject prefer the machine to real
life?
Nozick also believes that if pleasure were the only intrinsic
value, people would have an overriding reason to be hooked up to an
"experience machine", which would produce favorable sensations.
The argument
The argument is along these lines:
Premise 1: If experiencing as much pleasure as we can is all
that matters to us, then if we will experience more pleasure by doing x
than by doing y, we have no reason to do y rather than x.
Premise 2: We will experience more pleasure if we plug into the
experience machine than if we do not plug into the experience machine.
Conclusion 1: If experiencing as much pleasure as we can is all that
matters to us, then we have no reason not to plug into the experience
machine. (P1&P2)
Premise 3: We have reason not to plug into the experience machine.
Conclusion 2: Experiencing as much pleasure as we can is not all that matters to us. (C1&P3, by Modus tollens)
Reasons not to plug in
Nozick provides three reasons not to plug into the machine.
We want to do certain things, and not just have the experience of doing them.
"In the case of certain experiences, it is only because first we
want to do the actions that we want the experiences of doing them or
thinking we’ve done them."
We want to be a certain sort of person.
"Someone floating in a tank is an indeterminate blob...Is he
courageous, kind, intelligent, witty, loving? It’s not merely that it’s
difficult to tell; there’s no way he is. Plugging into the machine is a
form of suicide."
Plugging into an experience machine limits us to a man-made reality (it limits us to what we can make).
"There is no actual contact with any deeper reality, though the experience of it can be simulated."
Additionally
These are not quoted by Nozick himself, but rather other philosophers who have come up with or shared additional reasons.
Status Quo Bias, humans tend to dislike change, especially when considering the thought of having to be prodded with wires.
We would never see our real family and friends again, although unbeknownst to us.
The concept of free will becomes murky.
Previous experiences with technological failure; people don't trust machines.
Argument against hedonism
Hedonism
states that the things in life worth pursuing are the highest good, or
the things that will make one happiest both long term and short term.
Happiness is the highest value in human life. The Experience Machine is
hedonistic, and yet people still refuse to be plugged in for the reasons
listed above. Therefore, a conclusion is made that being personally
happy is not the greatest value everyone carries.
Counterarguments
Psychologist and philosopher Joshua Greene says that intuitions about the experience machine may be affected by status quo bias, and suggests reformulating the thought experiment in a form which counters this. According to his version:
you wake up in a plain white room. You are seated in a reclining
chair with a steel contraption on your head. A woman in a white coat is
standing over you. 'The year is 2659,' she explains, 'The life with
which you are familiar is an experience machine program selected by you
some forty years ago. We at IEM interrupt our client's programs at
ten-year intervals to ensure client satisfaction. Our records indicate
that at your three previous interruptions you deemed your program
satisfactory and chose to continue. As before, if you choose to continue
with your program you will return to your life as you know it with no
recollection of this interruption. Your friends, loved ones, and
projects will all be there. Of course, you may choose to terminate your
program at this point if you are unsatisfied for any reason. Do you
intend to continue with your program?
If a reader feels differently about this version of the story
compared to the form that Nozick offers, according to Greene, this is
due to status quo bias.
A similar counterargument was raised in a paper titled If You Like It, Does It Matter If It's Real?
by philosopher Felipe de Brigard. In contrast to the main experiment,
De Brigard asked 72 US university undergraduates whether they would like
to disconnect from the machine given that they were already in it.
About their "real" life, they were told one of three stories: (a)
nothing; (b) that they were prisoners in a maximum security prison; or
(c) that they were multimillionaire artists living in Monaco.
Of those who were told nothing of their "real" lives, 54% wished to
disconnect from the machine. Of those who were told they were prisoners,
only 13% wished to disconnect. This implies that one's real-life
quality impacts whether it is preferred to the machine. Of those told
they were rich inhabitants of Monaco, half chose to disconnect,
comparable to the proportion given no information about their "real"
life. De Brigard attributes his findings to status quo bias. He argues
that someone's decision not to step into the machine has more to do with
wanting the status quo than with preference of the current life over
the simulated one.
De Brigard points out that Nozick never empirically verified his
third premise. Nozick never tested his claims, arguing instead that it
must naturally be the case. Later philosophers and psychologists then
matched this with their own beliefs.
In fiction
Before
it became a philosophical thought experiment in the mid-seventies, the
pleasurable but simulated experience versus reality dilemma had been a
staple of science fiction; for example in the short story "The Chamber
of Life" by Green Peyton Wertenbaker [fr], published in the magazine Amazing Stories in October 1929. The 1996 novel Infinite Jest by David Foster Wallace involves a similar formulation of the experience machine. The novel revolves around a film titled Infinite Jest
that is lethally pleasurable: the film is so entertaining that, once
watched, the viewer will desire nothing else but to watch the film over
and over. Examples of movies centering on machines capable of replaying experiences previously recorded include the 1983 film Brainstorm and the 1995 film Strange Days.
The choice between standard human life and transforming into
creatures that can experience a much more intense pleasure life is also
one of the main twists of the classic novel City,
by Clifford Simak. In that story, as opposed to Nozick's argument, most
people opt for the pleasure life, mostly because they can fully
appreciate what they can gain in the process thanks to a sophisticated
language method, suggesting that the terms of the choice have to be well
chosen and fully understood for the experience to be significant.
It also is a running theme of the 1999 film The Matrix. Agent Smith's
account of the early history of the Matrix includes the idea that
humans reject a virtual reality that offers them paradise; however,
later his informant Cypher
is willing to betray his colleagues because he would prefer to be
reinserted into an (admittedly less perfect) Matrix as a wealthy and
successful man than continue to live in the harsh realities outside the
simulation. While this later version of the Matrix is not a
paradise-like reality in the literal sense, it may be argued that it is a
lot like a pleasure-inducing experience machine, since Cypher is given
the opportunity to have a prominent position of power and wealth in this
new simulation. As he says while dining at a simulated restaurant:
"You
know, I know this steak doesn't exist. I know that when I put it in my
mouth, the Matrix is telling my brain that it is juicy, and delicious.
After nine years, you know what I realize? Ignorance is bliss."
Another example of Nozick's experience machine would be the PASIV Device presented within Christopher Nolan's Inception.
A film that directly confronts the protagonist with the choice of an experience machine is Virtual Revolution.
The majority of the film's future population are the hedonists of the
experiment: 'Connected', that is having chosen a virtual existence over
their real one. The experiences are customized into 'verses with themes
much like modern video games (fantasy questing, first person shooting),
but upgraded via a brain-computer interface to send data to all five
senses and to block out true reality.
Gene silencing is often considered the same as gene knockdown.When genes are silenced, their expression is reduced. In contrast, when genes are knocked out, they are completely erased from the organism's genome and, thus, have no expression. Gene silencing is considered a gene knockdown mechanism since the methods used to silence genes, such as RNAi, CRISPR, or siRNA, generally reduce the expression of a gene by at least 70% but do not eliminate it. Methods using gene silencing are often considered better than gene knockouts since they allow researchers to study essential genes that are required for the animal models
to survive and cannot be removed. In addition, they provide a more
complete view on the development of diseases since diseases are
generally associated with genes that have a reduced expression.
Antisense oligonucleotides were discovered in 1978 by Paul Zamecnik and Mary Stephenson. Oligonucleotides, which are short nucleic acid fragments, bind to complementary target mRNA molecules when added to the cell. These molecules can be composed of single-stranded DNA or RNA and are generally 13–25 nucleotides long. The antisense oligonucleotides can affect gene expression in two ways: by using an RNase H-dependent mechanism or by using a steric blocking mechanism. RNase H-dependent oligonucleotides cause the target mRNA molecules to be degraded, while steric-blocker oligonucleotides prevent translation of the mRNA molecule.
The majority of antisense drugs function through the RNase H-dependent
mechanism, in which RNase H hydrolyzes the RNA strand of the DNA/RNA heteroduplex. expression.
Ribozymes
General mechanism utilized by ribozymes to cleave RNA molecules
Ribozymes are catalytic RNA molecules used to inhibit gene expression. These molecules work by cleaving mRNA molecules, essentially silencing the genes that produced them. Sidney Altman and Thomas Cech
first discovered catalytic RNA molecules, RNase P and group II intron
ribozymes, in 1989 and won the Nobel Prize for their discovery. Several types of ribozyme motifs exist, including hammerhead, hairpin, hepatitis delta virus, group I, group II, and RNase P ribozymes. Hammerhead, hairpin, and hepatitis delta virus (HDV) ribozyme motifs are generally found in viruses or viroid RNAs. These motifs are able to self-cleave a specific phosphodiester bond on an mRNA molecule. Lower eukaryotes and a few bacteria contain group I and group II ribozymes. These motifs can self-splice by cleaving and joining phosphodiester bonds. The last ribozyme motif, the RNase P ribozyme, is found in Escherichia coli and is known for its ability to cleave the phosphodiester bonds of several tRNA precursors when joined to a protein cofactor.
The general catalytic mechanism used by ribozymes is similar to the mechanism used by protein ribonucleases.[12]
These catalytic RNA molecules bind to a specific site and attack the
neighboring phosphate in the RNA backbone with their 2' oxygen, which
acts as a nucleophile, resulting in the formation of cleaved products with a 2'3'-cyclic phosphate and a 5' hydroxyl terminal end.
This catalytic mechanism has been increasingly used by scientists to
perform sequence-specific cleavage of target mRNA molecules. In
addition, attempts are being made to use ribozymes to produce gene
silencing therapeutics, which would silence genes that are responsible
for causing diseases.
RNA interference
Left:Overview of RNA interference.
RNA interference (RNAi) is a natural process used by cells to regulate gene expression. It was discovered in 1998 by Andrew Fire and Craig Mello, who won the Nobel Prize for their discovery in 2006. The process to silence genes first begins with the entrance of a double-stranded RNA (dsRNA) molecule into the cell, which triggers the RNAi pathway. The double-stranded molecule is then cut into small double-stranded fragments by an enzyme called Dicer. These small fragments, which include small interfering RNAs (siRNA) and microRNA (miRNA), are approximately 21–23 nucleotides in length. The fragments integrate into a multi-subunit protein called the RNA-induced silencing complex, which contains Argonaute proteins that are essential components of the RNAi pathway.
One strand of the molecule, called the "guide" strand, binds to RISC,
while the other strand, known as the "passenger" strand is degraded.
The guide or antisense strand of the fragment that remains bound to
RISC directs the sequence-specific silencing of the target mRNA
molecule.
The genes can be silenced by siRNA molecules that cause the
endonucleatic cleavage of the target mRNA molecules or by miRNA
molecules that suppress translation of the mRNA molecule.
With the cleavage or translational repression of the mRNA molecules,
the genes that form them are rendered essentially inactive. RNAi is thought to have evolved as a cellular defense mechanism against invaders, such as RNA viruses, or to combat the proliferation of transposons within a cell's DNA. Both RNA viruses and transposons can exist as double-stranded RNA and lead to the activation of RNAi. Currently, siRNAs are being widely used to suppress specific gene expression and to assess the function of genes. Companies utilizing this approach include Alnylam, Sanofi, Arrowhead, Discerna, and Persomics, among others.
The three prime untranslated regions (3'UTRs) of messenger RNAs (mRNAs) often contain regulatory sequences that post-transcriptionally cause gene silencing. Such 3'-UTRs often contain both binding sites for microRNAs (miRNAs) as well as for regulatory proteins. By binding to specific sites within the 3'-UTR, a large number of specific miRNAs decrease gene expression of their particular target mRNAs by either inhibiting translation or directly causing degradation of the transcript, using a mechanism similar to RNA interference (see MicroRNA). The 3'-UTR also may have silencer regions that bind repressor proteins that inhibit the expression of an mRNA.
The 3'-UTR often contains microRNA response elements (MREs).
MREs are sequences to which miRNAs bind and cause gene silencing.
These are prevalent motifs within 3'-UTRs. Among all regulatory motifs
within the 3'-UTRs (e.g. including silencer regions), MREs make up about
half of the motifs.
As of 2014, the miRBase web site, an archive of miRNA sequences
and annotations, listed 28,645 entries in 233 biologic species. Of
these, 1,881 miRNAs were in annotated human miRNA loci. miRNAs were
predicted to each have an average of about four hundred target mRNAs
(causing gene silencing of several hundred genes). Freidman et al. estimate that >45,000 miRNA target sites within human mRNA 3'UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs.
Direct experiments show that a single miRNA can reduce the stability of hundreds of unique mRNAs. Other experiments show that a single miRNA may repress the production of hundreds of proteins, but that this repression often is relatively mild (less than 2-fold).
The effects of miRNA dysregulation of gene expression seem to be important in cancer. For instance, in gastrointestinal cancers, nine miRNAs have been identified as epigenetically altered and effective in down regulating DNA repair enzymes.
The effects of miRNA dysregulation of gene expression also seem to be important in neuropsychiatric
disorders, such as schizophrenia, bipolar disorder, major depression,
Parkinson's disease, Alzheimer's disease and autism spectrum disorders.
RNA interference has been used to silence genes associated with several cancers. In in vitro studies of chronic myelogenous leukemia (CML), siRNA was used to cleave the fusion protein, BCR-ABL, which prevents the drug Gleevec (imatinib) from binding to the cancer cells. Cleaving the fusion protein reduced the amount of transformed hematopoietic cells that spread throughout the body by increasing the sensitivity of the cells to the drug.
RNA interference can also be used to target specific mutants. For
instance, siRNAs were able to bind specifically to tumor suppressor p53 molecules containing a single point mutation and destroy it, while leaving the wild-type suppressor intact.
Receptors involved in mitogenic pathways that lead to the increased production of cancer cells there have also been targeted by siRNA molecules. The chemokine receptor chemokine receptor 4 (CXCR4),
associated with the proliferation of breast cancer, was cleaved by
siRNA molecules that reduced the number of divisions commonly observed
by the cancer cells.
Researchers have also used siRNAs to selectively regulate the
expression of cancer-related genes. Antiapoptotic proteins, such as clusterin and survivin, are often expressed in cancer cells. Clusterin and survivin-targeting siRNAs were used to reduce the number
of antiapoptotic proteins and, thus, increase the sensitivity of the
cancer cells to chemotherapy treatments. In vivo
studies are also being increasingly utilized to study the potential use
of siRNA molecules in cancer therapeutics. For instance, mice implanted
with colon adenocarcinoma cells were found to survive longer when the cells were pretreated with siRNAs that targeted B-catenin in the cancer cells.
Infectious disease
Viruses
Viral
genes and host genes that are required for viruses to replicate or
enter the cell, or that play an important role in the life cycle of the
virus are often targeted by antiviral therapies. RNAi has been used to
target genes in several viral diseases, such as the human immunodeficiency virus (HIV) and hepatitis. In particular, siRNA was used to silence the primary HIV receptor chemokine receptor 5 (CCR5). This prevented the virus from entering the human peripheral blood lymphocytes and the primary hematopoietic stem cells. A similar technique was used to decrease the amount of the detectable virus in hepatitis B
and C infected cells. In hepatitis B, siRNA silencing was used to
target the surface antigen on the hepatitis B virus and led to a
decrease in the number of viral components. In addition, siRNA techniques used in hepatitis C were able to lower the amount of the virus in the cell by 98%.
RNA interference has been in commercial use to control virus diseases of plants for over 20 years (see Plant disease resistance).
In 1986–1990, multiple examples of "coat protein-mediated resistance"
against plant viruses were published, before RNAi had been discovered.
In 1993, work with tobacco etch virus first demonstrated that host
organisms can target specific virus or mRNA sequences for degradation,
and that this activity is the mechanism behind some examples of virus
resistance in transgenic plants.
The discovery of small interfering RNAs (the specificity determinant in
RNA-mediated gene silencing) also utilized virus-induced
post-transcriptional gene silencing in plants.
By 1994, transgenic squash varieties had been generated expressing coat
protein genes from three different viruses, providing squash hybrids
with field-validated multiviral resistance that remain in commercial use
at present. Potato lines expressing viral replicase sequences that
confer resistance to potato leafroll virus were sold under the trade
names NewLeaf Y and NewLeaf Plus, and were widely accepted in commercial
production in 1999–2001, until McDonald's Corp. decided not to purchase
GM potatoes and Monsanto decided to close their NatureMark potato business.
Another frequently cited example of virus resistance mediated by gene
silencing involves papaya, where the Hawaiian papaya industry was
rescued by virus-resistant GM papayas produced and licensed by
university researchers rather than a large corporation. These papayas also remain in use at present, although not without significant public protest, which is notably less evident in medical uses of gene silencing.
Gene silencing techniques have also been used to target other viruses, such as the human papilloma virus, the West Nile virus,
and the Tulane virus. The E6 gene in tumor samples retrieved from
patients with the human papilloma virus was targeted and found to cause
apoptosis in the infected cells.[50]
Plasmid siRNA expression vectors used to target the West Nile virus
were also able to prevent the replication of viruses in cell lines. In addition, siRNA has been found to be successful in preventing the replication of the Tulane virus, part of the virus family Caliciviridae, by targeting both its structural and non-structural genes.
By targeting the NTPase gene, one dose of siRNA 4 hours pre-infection
was shown to control Tulane virus replication for 48 hours
post-infection, reducing the viral titer by up to 2.6 logarithms.
Although the Tulane virus is species-specific and does not affect
humans, it has been shown to be closely related to the human norovirus, which is the most common cause of acute gastroenteritis and food-borne disease outbreaks in the United States.
Human noroviruses are notorious for being difficult to study in the
laboratory, but the Tulane virus offers a model through which to study
this family of viruses for the clinical goal of developing therapies
that can be used to treat illnesses caused by human norovirus.
Bacteria
Structure of a typical Gram-positive bacterial cell
Unlike viruses, bacteria are not as susceptible to silencing by siRNA.
This is largely due to how bacteria replicate. Bacteria replicate
outside of the host cell and do not contain the necessary machinery for
RNAi to function.
However, bacterial infections can still be suppressed by siRNA by
targeting the host genes that are involved in the immune response caused
by the infection or by targeting the host genes involved in mediating
the entry of bacteria into cells. For instance, siRNA was used to reduce the amount of pro-inflammatory cytokines expressed in the cells of mice treated with lipopolysaccharide (LPS). The reduced expression of the inflammatory cytokine, tumor necrosis factor α (TNFα), in turn, caused a reduction in the septic shock felt by the LPS-treated mice. In addition, siRNA was used to prevent the bacteria, Psueomonas aeruginosa, from invading murine lung epithelial cells by knocking down the caveolin-2 (CAV2) gene.
Thus, though bacteria cannot be directly targeted by siRNA mechanisms,
they can still be affected by siRNA when the components involved in the
bacterial infection are targeted.
Crystallographic structure of the N-terminal region of the human huntingtin protein.
Huntington's disease (HD) results from a mutation in the huntingtin gene that causes an excess of CAG repeats. The gene then forms a mutated huntingtin protein with polyglutamine repeats near the amino terminus. This disease is incurable and known to cause motor, cognitive, and behavioral deficits. Researchers have been looking to gene silencing as a potential therapeutic for HD.
Gene silencing can be used to treat HD by targeting the mutant
huntingtin protein. The mutant huntingtin protein has been targeted
through gene silencing that is allele specific using allele specific oligonucleotides. In this method, the antisense oligonucleotides are used to target single nucleotide polymorphism (SNPs),
which are single nucleotide changes in the DNA sequence, since HD
patients have been found to share common SNPs that are associated with
the mutated huntingtin allele. It has been found that approximately 85%
of patients with HD can be covered when three SNPs are targeted. In
addition, when antisense oligonucleotides were used to target an
HD-associated SNP in mice, there was a 50% decrease in the mutant
huntingtin protein.
Non-allele specific gene silencing using siRNA molecules has also
been used to silence the mutant huntingtin proteins. Through this
approach, instead of targeting SNPs on the mutated protein, all of the
normal and mutated huntingtin proteins are targeted. When studied in
mice, it was found that siRNA could reduce the normal and mutant
huntingtin levels by 75%. At this level, they found that the mice
developed improved motor control and a longer survival rate when compared to the controls. Thus, gene silencing methods may prove to be beneficial in treating HD.
Amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease, is a motor neuron disease that affects the brain and spinal cord. The disease causes motor neurons to degenerate, which eventually leads to neuron death and muscular degeneration. Hundreds of mutations in the Cu/Zn superoxide dismutase (SOD1) gene have been found to cause ALS. Gene silencing has been used to knock down the SOD1 mutant that is characteristic of ALS.
In specific, siRNA molecules have been successfully used to target the
SOD1 mutant gene and reduce its expression through allele-specific gene
silencing.
Therapeutics challenges
Basic mechanism used by viral vectors to deliver genes to target cells. Example shown is a lentiviral vector.
There are several challenges associated with gene silencing therapies, including delivery
and specificity for targeted cells. For instance, for treatment of
neurodegenerative disorders, molecules for a prospective gene silencing
therapy must be delivered to the brain. The blood–brain barrier
makes it difficult to deliver molecules into the brain through the
bloodstream by preventing the passage of the majority of molecules that
are injected or absorbed into the blood. Thus, researchers have found that they must directly inject the molecules or implant pumps that push them into the brain.
Once inside the brain, however, the molecules must move inside of
the targeted cells. In order to efficiently deliver siRNA molecules
into the cells, viral vectors can be used.
Nevertheless, this method of delivery can also be problematic as it
can elicit an immune response against the molecules. In addition to
delivery, specificity has also been found to be an issue in gene
silencing. Both antisense oligonucleotides and siRNA molecules can
potentially bind to the wrong mRNA molecule.
Thus, researchers are searching for more efficient methods to deliver
and develop specific gene silencing therapeutics that are still safe and
effective.
Arctic Apples are a suite of trademarked
apples that contain a nonbrowning trait created by using gene silencing
to reduce the expression of polyphenol oxidase (PPO). It is the first
approved food product to use this technique.
The magic bullet is a scientific concept developed by the German Nobel laureate Paul Ehrlich in 1907. While working at the Institute of Experimental Therapy (Institut für experimentelle Therapie), Ehrlich formed an idea that it could be possible to kill specific microbes (such as bacteria), which cause diseases in the body, without harming the body itself. He named the hypothetical agent as Zauberkugel, and used the English translation "magic bullet" in The Harben Lectures at London. The name itself is a reference to an old German myth about a bullet that cannot miss its target. Ehrlich had in mind Carl Maria von Weber's popular 1821 operaDer Freischütz, in which a young hunter is required to hit an impossible target in order to marry his bride.
Ehrlich envisioned that just like a bullet fired from a gun to
hit a specific target, there could be a way to specifically target
invading microbes. His continued research to discover the magic bullet
resulted in further knowledge of the functions of the body's immune system, and in the development of Salvarsan, the first effective drug for syphilis, in 1909. His works were the foundation of immunology, and for his contributions he shared the 1908 Nobel Prize in Physiology or Medicine with Élie Metchnikoff.
Ehrlich's discovery of Salvarsan in 1909 for the treatment of syphilis is termed as the first magic bullet. This led to the foundation of the concept of chemotherapy.
Background
Research on antibody
In the early 1890s, Paul Ehrlich started to work with Emil Behring, professor of medicine at the University of Marburg. Behring had been investigating antibacterial agents and discovered a diphtheriaantitoxin (that is, antibodies that target a biological toxin produced by the diphtheria bacteria Corynebacterium diphtheriae). (For that discovery, Bering was the first recipient of the Nobel Prize in Physiology or Medicine in 1901. Ehrlich was also nominated for that year.)
From Behring's work, Ehrlich understood that antibodies produced in the
blood could attack invading pathogens without any harmful effect on the
body. He speculated that these antibodies act as bullets fired from a
gun to target specific microbes. But after further research, he realised
that antibodies sometimes failed to kill microbes. This led him to
abandon his first concept of the magic bullet.
Research on therapeutic properties of dyes
Ehrlich joined the Institute of Experimental Therapy (Institut für experimentelle Therapie) at Frankfurt am Main, Germany,
in 1899, becoming the director of its research institute the
Georg–Speyer Haus in 1906. Here his research focused on testing
arsenical dyes for killing microbes. Arsenic was an infamous poison, and
his attempt was criticised. He was publicly lampooned as an imaginary
"Dr Phantasus".
But Ehrlich's rationale was that the chemical structure called side
chain forms antibodies that bind to toxins (such as pathogens and their
products); similarly, chemical dyes such as arsenic compounds could also
produce such side chains to kill the same microbes. This led him to
propose a new concept called "side-chain theory".
(Later in 1900, he revised his concept as "receptor theory".) Based on
his new theory, he postulated that in order to kill microbes, "wir müssen chemisch zielen lernen" ("we have to learn how to aim chemically").
His institute was convenient as it was adjacent to a dye factory.
He began testing a number of compounds against different microbes. It
was during his research that he coined the terms "chemotherapy" and "magic bullet". Although he used the German word zauberkugel
in his earlier writings, the first time he introduced the English term
"magic bullet" was at a Harben Lecture in London in 1908. By 1901, with the help of Japanese microbiologist Kiyoshi Shiga, Ehrlich experimented with hundreds of dyes on mice infected with trypanosome, a protozoan parasite that causes sleeping sickness. In 1904 they successfully prepared a red azo dye they called Trypan Red for the treatment of sleeping sickness.
Discovery of the first magic bullet – Salvarsan
In
1906 Ehrlich developed a new derivative of arsenic compound, which he
code-named Compound 606 (the number representing the series of all his
tested compounds). The compound was effective against malaria infection
in experimental animals. In 1905, Fritz Schaudinn and Erich Hoffmann identified a spirochaete bacterium (Treponema pallidum) as the causative organism of syphilis. With this new knowledge, Ehrlich tested Compound 606 (chemically arsphenamine) on a syphilis-infected rabbit. He did not recognise its effectiveness. Sahachiro Hata
went over Ehrlich's work and found on 31 August 1909 that the rabbit,
which had been injected with Compound 606, was cured using only a single
dose, the rabbit showing no adverse effect.
The normal treatment procedure of syphilis at the time involved
two to four years routine injection with mercury. Ehrlich, after
receiving this information, performed experiments on human patients with
the same success. After convincing clinical trials, the compound number
606 was given the trade name "Salvarsan", a portmanteau for "saving
arsenic".
Salvarsan was commercially introduced in 1910, and in 1913, a less
toxic form, "Neosalvarsan" (Compound 914), was released in the market.
These drugs became the principal treatments of syphilis until the
arrival of penicillin and other novel antibiotics towards the middle of
the 20th century.
Ehrlich created the concept of magic bullet based on the
development of arsphenamine and introduced the English phrase "magic
bullet" in The Harben Lectures for 1907 of the Royal Institute of Public Health at London. However, he had used the German word Zauberkugel in his earlier works on the side-chain theory. The magic bullet became the foundation of modern pharmaceutical research.
A colored electron microscopy image of methicillin-resistant staphylococcus aureus (MRSA), a bacterium commonly targeted by broad-spectrum antibiotics
A broad-spectrum antibiotic is an antibiotic that acts on the two major bacterial groups, Gram-positive and Gram-negative, or any antibiotic that acts against a wide range of disease-causing bacteria. These medications are used when a bacterial infection is suspected but the group of bacteria is unknown (also called empiric therapy) or when infection with multiple groups of bacteria is suspected. This is in contrast to a narrow-spectrum antibiotic, which is effective against only a specific group of bacteria.
Although powerful, broad-spectrum antibiotics pose specific risks,
particularly the disruption of native, normal bacteria and the
development of antimicrobial resistance. An example of a commonly used broad-spectrum antibiotic is ampicillin.
Bacterial targets
Antibiotics
are often grouped by their ability to act on different bacterial
groups. Although bacteria are biologically classified using taxonomy,
disease-causing bacteria have historically been classified by their
microscopic appearance and chemical function. The morphology of the
organism may be classified as cocci, diplococci, bacilli
(also known as "rods"), spiral-shaped or pleomorphic. Additional
classification occurs through the organism's ability to take up the Gram stain and counter-stain;
bacteria that take up the crystal violet dye stain are referred to as
"gram-positive," those that take up the counterstain only are
"gram-negative," and those that remain unstained are referred to as
"atypical." Further classification includes their requirement for
oxygen (i.e., aerobic or anaerobic), patterns of hemolysis,
or other chemical properties. The most commonly encountered groupings
of bacteria include gram-positive cocci, gram-negative bacilli, atypical
bacteria, and anaerobic bacteria.
Simplified diagram showing common disease-causing bacteria and the antibiotics which act against them.
Empiric antibiotic therapy refers to the use of antibiotics to treat a
suspected bacterial infection despite lack of a specific bacterial
diagnosis. Definitive diagnosis of the species of bacteria often occurs
through culture of blood, sputum, or urine, and can be delayed by 24 to 72 hours. Antibiotics are generally given after
the culture specimen has been taken from the patient in order to
preserve the bacteria in the specimen and ensure accurate diagnosis. Alternatively, some species may be identified through a urine or stool test.
There are an estimated 38 trillion microorganisms that colonize the human body. As a side-effect of therapy, antibiotics can change the body's normal microbial content
by attacking indiscriminately both the pathological and naturally
occurring, beneficial or harmless bacteria found in the intestines,
lungs and bladder. The destruction of the body's normal bacterial flora is thought to disrupt immunity, nutrition, and lead to a relative overgrowth in some bacteria or fungi. An overgrowth of drug-resistant microorganisms can lead to a secondary infection such as Clostridioides difficile ("C. diff") or candidiasis ("thrush").
This side-effect is more likely with the use of broad-spectrum
antibiotics, given their greater potential to disrupt a larger variety
of normal human flora. The use of doxycycline in acne vulgaris has been associated with increased risk of Crohn's disease, although a later study indicated a link between acne vulgaris and IBS irrespective of the use of antibiotics. Likewise, the use of minocycline in acne vulgaris has been associated with skin and gut dysbiosis.
Action of β-lactamase and decarboxylation of the intermediate
Core structure of penicillins (top) and cephalosporins (bottom). Beta-lactam ring in red.Escherichia coli
bacteria on the right are sensitive to two beta-lactam antibiotics, and
do not grow in the semi-circular regions surrounding antibiotics. E. coli
bacteria on the left are resistant to beta-lactam antibiotics, and grow
next to one antibiotic (bottom) and are less inhibited by another
antibiotic (top).
Beta-lactamases produced by gram-negative bacteria are usually secreted, especially when antibiotics are present in the environment.
Structure
The structure of a Streptomyces serine β-lactamase (SBLs) is given by 1BSG. The alpha-beta fold (InterPro: IPR012338) resembles that of a DD-transpeptidase, from which the enzyme is thought to have evolved. β-lactam antibiotics bind to DD-transpeptidases
to inhibit bacterial cell wall biosynthesis. Serine β-lactamases are
grouped by sequence similarity into types A, C, and D.
The other type of beta-lactamase is of the metallo type ("type B"). Metallo-beta-lactamases (MBLs) need metal ion(s) (1 or 2 Zn2+ ions) on their active site for their catalytic activities. The structure of the New Delhi metallo-beta-lactamase 1 is given by 6C89. It resembles a RNase Z, from which it is thought to have evolved.
Mechanism of action
The two types of beta-lactamases work on the basis of the two basic mechanisms of opening the β-lactam ring.
The SBLs are similar in structure and mechanistically to the
β-lactam target penicillin-binding proteins (PBPs) which are necessary
for cell wall building and modifying. SBLs and PBPs both covalently
change an active site serine residue. The difference between the PBPs
and SBLs is that the latter generates free enzyme and inactive
antibiotic by the very quick hydrolysis of the acyl-enzyme intermediate.
The MBLs use the Zn2+ ions to activate a binding site
water molecule for the hydrolysis of the β-lactam ring. Zinc chelators
have recently been investigated as metallo-β-lactamase inhibitors, as
they are often able to restore carbapenem susceptibility.
Penicillinase
Penicillinase is a specific type of β-lactamase, showing specificity for penicillins, again by hydrolysing the β-lactam ring. Molecular weights of the various penicillinases tend to cluster near 50 kilodaltons.
Penicillinase was the first β-lactamase to be identified. It was first isolated by Abraham and Chain in 1940 from E. coli (which are gram-negative) even before penicillin entered clinical use,
but penicillinase production quickly spread to bacteria that previously
did not produce it or produced it only rarely. Penicillinase-resistant
beta-lactams such as methicillin were developed, but there is now widespread resistance to even these.
Resistance in gram-negative bacteria
Among gram-negative bacteria, the emergence of resistance to
extended-spectrum cephalosporins has been a major concern. It appeared
initially in a limited number of bacterial species (E. cloacae, C. freundii, S. marcescens, and P. aeruginosa)
that could mutate to hyperproduce their chromosomal class C
β-lactamase. A few years later, resistance appeared in bacterial species
not naturally producing AmpC enzymes (K. pneumoniae, Salmonella spp., P. mirabilis)
due to the production of TEM- or SHV-type ESBLs (extended spectrum beta
lactamases). Characteristically, such resistance has included oxyimino-
(for example ceftizoxime, cefotaxime, ceftriaxone, and ceftazidime, as well as the oxyimino-monobactam aztreonam), but not 7-alpha-methoxy-cephalosporins (cephamycins; in other words, cefoxitin and cefotetan); has been blocked by inhibitors such as clavulanate, sulbactam or tazobactam and did not involve carbapenems and temocillin.
Chromosomal-mediated AmpC β-lactamases represent a new threat, since
they confer resistance to 7-alpha-methoxy-cephalosporins (cephamycins) such as cefoxitin or cefotetan
but are not affected by commercially available β-lactamase inhibitors,
and can, in strains with loss of outer membrane porins, provide
resistance to carbapenems.
Extended-spectrum beta-lactamase (ESBL)
Members of this family commonly express β-lactamases (e.g., TEM-3, TEM-4, and SHV-2 )
which confer resistance to expanded-spectrum (extended-spectrum)
cephalosporins. In the mid-1980s, this new group of enzymes, the
extended-spectrum β-lactamases (ESBLs), was detected (first detected in
1979). The prevalence of ESBL-producing bacteria have been gradually increasing in acute care hospitals. The prevalence in the general population varies between countries, e.g. approximately 6% in Germany and France, 13% in Saudi Arabia, and 63% in Egypt.
ESBLs are beta-lactamases that hydrolyze extended-spectrum
cephalosporins with an oxyimino side chain. These cephalosporins include
cefotaxime, ceftriaxone, and ceftazidime, as well as the oxyimino-monobactam aztreonam. Thus ESBLs confer multi-resistance
to these antibiotics and related oxyimino-beta lactams. In typical
circumstances, they derive from genes for TEM-1, TEM-2, or SHV-1 by
mutations that alter the amino acid configuration around the active site
of these β-lactamases. A broader set of β-lactam antibiotics are
susceptible to hydrolysis by these enzymes. An increasing number of
ESBLs not of TEM or SHV lineage have recently been described.
The ESBLs are frequently plasmid encoded. Plasmids responsible for ESBL
production frequently carry genes encoding resistance to other drug
classes (for example, aminoglycosides). Therefore, antibiotic options in
the treatment of ESBL-producing organisms are extremely limited. Carbapenems are the treatment of choice for serious infections due to ESBL-producing organisms, yet carbapenem-resistant (primarily ertapenem-resistant) isolates have recently been reported. ESBL-producing organisms may appear susceptible to some extended-spectrum cephalosporins. However, treatment with such antibiotics has been associated with high failure rates.
Types
"Amp resistance" redirects here. For resistance to antimicrobial peptides, see AMP resistance.
TEM beta-lactamases (class A)
TEM-1 is the most commonly encountered beta-lactamase in gram-negative bacteria. Up to 90% of ampicillin resistance in E. coli is due to the production of TEM-1. Also responsible for the ampicillin and penicillin resistance that is seen in H. influenzae and N. gonorrhoeae in increasing numbers. Although TEM-type beta-lactamases are most often found in E. coli and K. pneumoniae,
they are also found in other species of gram-negative bacteria with
increasing frequency. The amino acid substitutions responsible for the extended-spectrum beta lactamase (ESBL)
phenotype cluster around the active site of the enzyme and change its
configuration, allowing access to oxyimino-beta-lactam substrates.
Opening the active site to beta-lactam substrates also typically
enhances the susceptibility of the enzyme to β-lactamase inhibitors,
such as clavulanic acid. Single amino acid substitutions at positions
104, 164, 238, and 240 produce the ESBL phenotype, but ESBLs with the
broadest spectrum usually have more than a single amino acid
substitution. Based upon different combinations of changes, currently
140 TEM-type enzymes have been described. TEM-10, TEM-12, and TEM-26 are
among the most common in the United States.The term TEM comes from the name of the Athenian patient (Temoniera) from which the isolate was recovered in 1963.
SHV beta-lactamases (class A)
SHV-1
shares 68 percent of its amino acids with TEM-1 and has a similar
overall structure. The SHV-1 beta-lactamase is most commonly found in K. pneumoniae
and is responsible for up to 20% of the plasmid-mediated ampicillin
resistance in this species. ESBLs in this family also have amino acid
changes around the active site, most commonly at positions 238 or 238
and 240. More than 60 SHV varieties are known. SHV-5 and SHV-12 are
among the most common. The initials stand for "sulfhydryl reagent variable".
CTX-M beta-lactamases (class A)
These enzymes were named for their greater activity against cefotaxime than other oxyimino-beta-lactam substrates (e.g., ceftazidime, ceftriaxone, or cefepime).
Rather than arising by mutation, they represent examples of plasmid
acquisition of beta-lactamase genes normally found on the chromosome of Kluyvera
species, a group of rarely pathogenic commensal organisms. These
enzymes are not very closely related to TEM or SHV beta-lactamases in
that they show only approximately 40% identity with these two commonly
isolated beta-lactamases. More than 172 CTX-M enzymes are currently known. Despite their name, a few are more active on ceftazidime than cefotaxime. They are widely described among species of Enterobacteriaceae, mainly E. coli and K. pneumoniae.
Detected in the 1980s they have since the early 2000s spread and are
the now the predominant ESBL type in the world. They are generally
clustred into five groups based on sequencing homologies; CTX-M-1,
CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25. CTX-M-15 (belonging to the
CTX-M-1 cluster) is the most prevalent CTX-M-gene. An example of beta-lactamase CTX-M-15, along with ISEcp1, has been found to have transposed onto the chromosome of Klebsiella pneumoniae ATCC BAA-2146. The initials stand for "Cefotaxime-Munich".
OXA beta-lactamases (class D)
OXA beta-lactamases were long recognized as a less common but also plasmid-mediated beta-lactamase variety that could hydrolyze oxacillin
and related anti-staphylococcal penicillins. These beta-lactamases
differ from the TEM and SHV enzymes in that they belong to molecular
class D and functional group 2d. The OXA-type beta-lactamases confer
resistance to ampicillin and cephalothin and are characterized by their high hydrolytic activity against oxacillin and cloxacillin and the fact that they are poorly inhibited by clavulanic acid. Amino acid substitutions in OXA enzymes can also give the ESBL phenotype. While most ESBLs have been found in E. coli, K. pneumoniae, and other Enterobacteriaceae, the OXA-type ESBLs have been found mainly in P. aeruginosa. OXA-type ESBLs have been found mainly in Pseudomonas aeruginosa
isolates from Turkey and France. The OXA beta-lactamase family was
originally created as a phenotypic rather than a genotypic group for a
few beta-lactamases that had a specific hydrolysis profile. Therefore,
there is as little as 20% sequence homology among some of the members of
this family. However, recent additions to this family show some degree
of homology to one or more of the existing members of the OXA
beta-lactamase family. Some confer resistance predominantly to
ceftazidime, but OXA-17 confers greater resistance to cefotaxime and
cefepime than it does resistance to ceftazidime.
Others
Other
plasmid-mediated ESBLs, such as PER, VEB, GES, and IBC beta-lactamases,
have been described but are uncommon and have been found mainly in P. aeruginosa
and at a limited number of geographic sites. PER-1 in isolates in
Turkey, France, and Italy; VEB-1 and VEB-2 in strains from Southeast
Asia; and GES-1, GES-2, and IBC-2 in isolates from South Africa, France,
and Greece. PER-1 is also common in multiresistant acinetobacter
species in Korea and Turkey. Some of these enzymes are found in
Enterobacteriaceae as well, whereas other uncommon ESBLs (such as BES-1,
IBC-1, SFO-1, and TLA-1) have been found only in Enterobacteriaceae.
Treatment
While
ESBL-producing organisms were previously associated with hospitals and
institutional care, these organisms are now increasingly found in the
community. CTX-M-15-positive E. coli are a cause of community-acquired urinary infections in the UK, and tend to be resistant to all oral β-lactam antibiotics, as well as quinolones and sulfonamides. Treatment options may include nitrofurantoin, fosfomycin, mecillinam and chloramphenicol. In desperation, once-daily ertapenem or gentamicin injections may also be used.
Inhibitor-resistant β-lactamases
Although
the inhibitor-resistant β-lactamases are not ESBLs, they are often
discussed with ESBLs because they are also derivatives of the classical
TEM- or SHV-type enzymes. These enzymes were at first given the
designation IRT for inhibitor-resistant TEM β-lactamase; however, all
have subsequently been renamed with numerical TEM designations. There
are at least 19 distinct inhibitor-resistant TEM β-lactamases.
Inhibitor-resistant TEM β-lactamases have been found mainly in clinical
isolates of E. coli, but also some strains of K. pneumoniae, Klebsiella oxytoca, P. mirabilis, and Citrobacter freundii. Although the inhibitor-resistant TEM variants are resistant to inhibition by clavulanic acid and sulbactam, thereby showing clinical resistance to the beta-lactam—lactamase inhibitor combinations of amoxicillin-clavulanate (co-amoxiclav), ticarcillin-clavulanate (co-ticarclav), and ampicillin/sulbactam, they normally remain susceptible to inhibition by tazobactam and subsequently the combination of piperacillin/tazobactam,
although resistance has been described. This is no longer a primarily
European epidemiology, it is found in northern parts of America often
and should be tested for with complex UTI's.
AmpC-type β-lactamases (class C)
AmpC
type β-lactamases are commonly isolated from extended-spectrum
cephalosporin-resistant gram-negative bacteria. AmpC β-lactamases (also
termed class C or group 1) are typically encoded on the chromosome of
many gram-negative bacteria including Citrobacter, Serratia and Enterobacter species where its expression is usually inducible; it may also occur on Escherichia coli but is not usually inducible, although it can be hyperexpressed. AmpC type β-lactamases may also be carried on plasmids.
AmpC β-lactamases, in contrast to ESBLs, hydrolyse broad and
extended-spectrum cephalosporins (cephamycins as well as to
oxyimino-β-lactams) but are not typically inhibited by the β-lactamase
inhibitors clavulanic acid and tazobactam, whereas avibactam can maintain inhibitory activity against this class of β-lactamases. AmpC-type β-lactamase organisms are often clinically grouped through the acronym, "SPACE": Serratia, Pseudomonas or Proteus, Acinetobacter, Citrobacter, and Enterobacter.
Carbapenemases
Carbapenems
are famously stable to AmpC β-lactamases and
extended-spectrum-β-lactamases. Carbapenemases are a diverse group of
β-lactamases that are active not only against the
oxyimino-cephalosporins and cephamycins but also against the
carbapenems. Aztreonam is stable to the metallo-β-lactamases,
but many IMP and VIM producers are resistant, owing to other mechanisms.
Carbapenemases were formerly believed to derive only from classes A,
B, and D, but a class C carbapenemase has been described.
IMP-type carbapenemases (metallo-β-lactamases) (class B)
Plasmid-mediated
IMP-type carbapenemases (IMP stands for active-on-imipenem), 19
varieties of which are currently known, became established in Japan in
the 1990s both in enteric gram-negative organisms and in Pseudomonas and Acinetobacter
species. IMP enzymes spread slowly to other countries in the Far East,
were reported from Europe in 1997, and have been found in Canada and
Brazil.
VIM (Verona integron-encoded metallo-β-lactamase) (Class B)
A
second growing family of carbapenemases, the VIM family, was reported
from Italy in 1999 and now includes 10 members, which have a wide
geographic distribution in Europe, South America, and the Far East and
have been found in the United States. VIM-1 was discovered in P. aeruginosa
in Italy in 1996; since then, VIM-2 - now the predominant variant - was
found repeatedly in Europe and the Far East; VIM-3 and -4 are minor
variants of VIM-2 and -1, respectively.
Amino acid sequence diversity is up to 10% in the VIM family, 15%
in the IMP family, and 70% between VIM and IMP. Enzymes of both the
families, nevertheless, are similar. Both are integron-associated,
sometimes within plasmids. Both hydrolyse all β-lactams except
monobactams, and evade all β-lactam inhibitors. The VIM enzymes are
among the most widely distributed MBLs, with >40 VIM variants having
been reported. Biochemical and biophysical studies revealed that VIM
variants have only small variations in their kinetic parameters but
substantial differences in their thermal stabilities and inhibition
profiles.
OXA (oxacillinase) group of β-lactamases (class D)
The
OXA group of β-lactamases occur mainly in Acinetobacter species and are
divided into two clusters. OXA carbapenemases hydrolyse carbapenems
very slowly in vitro, and the high MICs seen for some
Acinetobacter hosts (>64 mg/L) may reflect secondary mechanisms. They
are sometimes augmented in clinical isolates by additional resistance
mechanisms, such as impermeability or efflux. OXA carbapenemases also
tend to have a reduced hydrolytic efficiency towards penicillins and
cephalosporins.
KPC (K. pneumoniae carbapenemase) (class A)
A
few class A enzymes, most noted the plasmid-mediated KPC enzymes, are
effective carbapenemases as well. Ten variants, KPC-2 through KPC-11 are
known, and they are distinguished by one or two amino acid
substitutions (KPC-1 was re-sequenced in 2008 and found to be 100%
homologous to published sequences of KPC-2). KPC-1 was found in North
Carolina, KPC-2 in Baltimore and KPC-3 in New York. They have only 45%
homology with SME and NMC/IMI enzymes and, unlike them, can be encoded
by self-transmissible plasmids.
As of February 2009, the class A Klebsiella pneumoniae carbapenemase (KPC) globally has been the most common carbapenemase, and was first detected in 1996 in North Carolina, USA. A 2010 publication indicated that KPC producing Enterobacteriaceae were becoming common in the United States.
CMY (class C)
The first class C carbapenemase was described in 2006 and was isolated from a virulent strain of Enterobacter aerogenes. It is carried on a plasmid, pYMG-1, and is therefore transmissible to other bacterial strains.
SME (Serratia marcescens enzymes), IMI (IMIpenem-hydrolysing β-lactamase), NMC and CcrA
In general, these are of little clinical significance.
CcrA (CfiA). Its gene occurs in ca. 1–3% of B. fragilis
isolates, but fewer produce the enzyme since expression demands
appropriate migration of an insertion sequence. CcrA was known before
imipenem was introduced, and producers have shown little subsequent
increase.
Originally described from New Delhi in 2009, this gene is now widespread in Escherichia coli and Klebsiella pneumoniae
from India and Pakistan. As of mid-2010, NDM-1 carrying bacteria have
been introduced to other countries (including the United States and UK),
most probably due to the large number of tourists travelling the globe,
who may have picked up the strain from the environment, as strains
containing the NDM-1 gene have been found in environmental samples in
India. NDM have several variants which share different properties.
Treatment of ESBL/AmpC/carbapenemases
General overview
In general, an isolate is suspected to be an ESBL producer when it shows in vitro susceptibility to the cephamycins (cefoxitin, cefotetan) but resistance to the third-generation cephalosporins and to aztreonam.
Moreover, one should suspect these strains when treatment with these
agents for gram-negative infections fails despite reported in vitro
susceptibility. Once an ESBL-producing strain is detected, the
laboratory should report it as "resistant" to all penicillins,
cephalosporins, and aztreonam, even if it is tested (in vitro) as
susceptible. Associated resistance to aminoglycosides and trimethoprim-sulfamethoxazole, as well as high frequency of co-existence of fluoroquinolone resistance, creates problems. Beta-lactamase inhibitors such as clavulanate, sulbactam, and tazobactamin vitro
inhibit most ESBLs, but the clinical effectiveness of
beta-lactam/beta-lactamase inhibitor combinations cannot be relied on
consistently for therapy. Cephamycins (cefoxitin and cefotetan)
are not hydrolyzed by majority of ESBLs, but are hydrolyzed by
associated AmpC-type β-lactamase. Also, β-lactam/β-lactamase inhibitor
combinations may not be effective against organisms that produce
AmpC-type β-lactamase. Sometimes these strains decrease the expression
of outer membrane proteins, rendering them resistant to cephamycins. In vivo studies have yielded mixed results against ESBL-producing K. pneumoniae. (Cefepime, a fourth-generation cephalosporin, has demonstrated in vitro stability in the presence of many ESBL/AmpC strains.) Currently, carbapenems
are, in general, regarded as the preferred agent for treatment of
infections due to ESBL-producing organisms. Carbapenems are resistant to
ESBL-mediated hydrolysis and exhibit excellent in vitro activity against strains of Enterobacteriaceae expressing ESBLs.
For organisms producing TEM and SHV type ESBLs, apparent in vitro sensitivity to cefepime and to piperacillin/tazobactam
is common, but both drugs show an inoculum effect, with diminished
susceptibility as the size of the inoculum is increased from 105 to 107 organisms.
Strains with some CTX-M–type and OXA-type ESBLs are resistant to cefepime on testing, despite the use of a standard inoculum.
AmpC-producing strains are typically resistant to oxyimino-beta lactams and to cephamycins and are susceptible to carbapenems; however, diminished porin expression can make such a strain carbapenem-resistant as well.
Carbapenemases
Strains with IMP-, VIM-, and OXA-type
carbapenemases usually remain susceptible. Resistance to
non-beta-lactam antibiotics is common in strains making any of these
enzymes, such that alternative options for non-beta-lactam therapy need
to be determined by direct susceptibility testing. Resistance to fluoroquinolones and aminoglycosides is especially high.
According to species
Escherichia coli or Klebsiella
For infections caused by ESBL-producing Escherichia coli or Klebsiella species, treatment with imipenem or meropenem has been associated with the best outcomes in terms of survival and bacteriologic clearance. Cefepime and piperacillin/tazobactam have been less successful. Ceftriaxone, cefotaxime, and ceftazidime have failed even more often, despite the organism's susceptibility to the antibiotic in vitro. Several reports have documented failure of cephamycin therapy as a result of resistance due to porin loss. Some patients have responded to aminoglycoside or quinolone therapy, but, in a recent comparison of ciprofloxacin and imipenem for bacteremia involving an ESBL-producing K. pneumoniae, imipenem produced the better outcome
Pseudomonas aeruginosa
There have been few clinical studies to define the optimal therapy for infections caused by ESBL producing Pseudomonas aeruginosa strains.
Use as a pharmaceutical
In
1957, amid concern about allergic reactions to penicillin-containing
antibiotics, a beta-lactamase was sold as an antidote under the brand
name neutrapen. It was theorized that the breakdown of penicillin by the enzyme would treat the allergic reaction. While it was not useful in acute anaphylactic shock, it showed positive results in cases of urticaria and joint pain suspected to be caused by penicillin allergy.
Its use was proposed in pediatric cases where penicillin allergy was
discovered upon administration of the polio vaccine, which used
penicillin as a preservative. However, some patients developed allergies to neutrapen. The Albany Hospital removed it from its formulary in 1960, only two years after adding it, citing lack of use. Some researchers continued to use it in experiments on penicillin resistance as late as 1972. It was voluntarily withdrawn from the American market by 3M Pharmaceuticals in 1997.
Detection
Beta-lactamase enzymatic activity can be detected using nitrocefin, a chromogenic cephalosporin substrate which changes color from yellow to red upon beta-lactamase mediated hydrolysis.
Extended spectrum beta lactamase (ESBL) screening can be
performed using disk-diffusion. Cefpodoxime, ceftazidime, aztreonam,
cefotaxime, and/or ceftriaxone discs are used.
Evolution
Beta-lactamases are ancient bacterial enzymes. Metallo β-lactamases ("class B") are all structurally similar to RNase Z and may have evolved from it. Of the three subclasses B1, B2, and B3, B1 and B2 are theorized to have evolved about one billion years ago,
while B3 seems to have arisen independently, possibly before the
divergence of the gram-positive and gram-negative eubacteria about two
billion years ago.
PNGM-1 (Papua New Guinea Metallo-β-lactamase-1) has both
metallo-β-lactamase (MBL) and tRNase Z activities, suggesting that
PNGM-1 is thought to have evolved from a tRNase Z, and that the B3 MBL
activity of PNGM-1 is a promiscuous activity and subclass B3 MBLs are
thought to have evolved through PNGM-1 activity. Subclasses B1 and B3 has been further subdivided.
Serine beta-lactamases (classes A, C, and D) appear to have evolved from DD-transpeptidases, which are penicillin-binding proteins involved in cell wall biosynthesis, and as such are one of the main targets of beta-lactam antibiotics.
These three classes show undetectable sequence similarity with each
other, but can still be compared using structural homology. Groups A and
D are sister taxa and group C diverged before A and D.
These serine-based enzymes, like the group B betalactamases, are of
ancient origin and are theorized to have evolved about two billion years
ago.
The OXA group (in class D) in particular is theorized to have
evolved on chromosomes and moved to plasmids on at least two separate
occasions.
Etymology
The "β" (beta) refers to the nitrogen's position on the second carbon in the ring. Lactam is a blend of lactone (from the Latinlactis, milk, since lactic acid was isolated from soured milk) and amide. The suffix -ase, indicating an enzyme, is derived from diastase (from the Greekdiastasis, "separation"), the first enzyme discovered in 1833 by Payen and Persoz.
