Fluoroquinolones: Mechanisms of Action and Resistance

In this animation, we demonstrate the biology of DNA replication leading to bacterial cell division in a gram positive bacterium, such as S. pneumoniae. The DNA is shown as a circular double strand within the bacterial cell. Like the DNA of all living organisms, it contains the unique genetic code for all of the proteins required for bacterial survival. Bacteria replicate by a process known as binary fission whereby one bacterium separates into 2 new daughter cells. However, before this can occur, the bacterium must make an identical copy of its complete circular DNA.

 In this animation, we demonstrate the biology of DNA replication leading to bacterial cell division in a gram positive bacterium, such as S. pneumoniae. The DNA is shown as a circular double strand within the bacterial cell. Like the DNA of all living organisms, it contains the unique genetic code for all of the proteins required for bacterial survival. Bacteria replicate by a process known as binary fission whereby one bacterium separates into 2 new daughter cells. However, before this can occur, the bacterium must make an identical copy of its complete circular DNA. DNA replication requires that the two strands of DNA separate so that the genetic code of the bacterium can be read and a new complimentary strand can be created for each of the original strands. To accomplish this, various enzymes known as helicases break the hydrogen bonds between the bases in the two DNA strands, unwind the strands from each other, and stabilize the exposed single strands, preventing them from joining back together. The points at which the two strands of DNA separate to allow replication of DNA are known as replication forks. The enzymes DNA polymerase then move along each strand of DNA, behind each replication fork synthesizing new DNA strands (in red) complementary to the original ones. As the replication forks move forward, positive superhelical twists in the DNA begin to accumulate ahead of them. In order for DNA replication to continue, these superhelical twists must be removed. The bacterial enzyme, DNA gyrase, which is also known as topoisomerase II, is responsible for removing the positive superhelical twists so that DNA replications can procede. DNA gyrase is an essential bacterial enzyme composed of two A and two B subunits which are products of the gyrA and gyrB genes. This enzyme has other important functions which affect the initiation of DNA replication and transcription of many genes. With the combined involvement of these enzymes, an entire duplicate copy of the bacterial genome is produced as the 2 replication forks move in opposite directions around the circular DNA genome. Eventually, as the 2 replication forks meet, 2 new complete chromosomes have been made, each consisting of 1 old and 1 new strand of DNA. This is referred to as semi-conservative replication. In order to allow the 2 new interlinked chromosomes to come apart, another bacterial enzyme is needed which is known as topoisomerase IV. This enzyme is structurally related to DNA gyrase and is coded for by the parC and parE genes. Topoisomerase IV allows for the 2 new inter-linked chromosomes to separate so that they can be segregated into 2 new daughter bacterial cells. Complete separation of bacterial cells Fluoroquinolones. First mechanism of action -- inhibition of DNA gyrase. Fluoroquinolones act by inhibiting the activity of both the DNA gyrase and the topoisomerase IV enzymes. For most gram negative bacteria, DNA gyrase is the primary fluoroquinolone target. Fluoroquinolones have been shown to bind specifically to the complex of DNA gyrase and DNA rather than to DNA gyrase alone. As a result of this binding, quinolones appear to stabilize the enzyme-DNA complexes which in turn results in breaks in the DNA that are fatal to the bacterium. A second mechanism of fluoroquinolone action is shown here. With some exceptions, topoisomerase IV is the primary target of fluoroquinolone action in most gram positive bacteria such as Staphylococci and Streptococci, with DNA gyrase being a secondary target. The separation of 2 new interlinked daughter strands of circular DNA is disrupted. The final result on the bacteria, however, is the same. Bacterial replication is disrupted and the bacterium breaks apart. The relative potency of different fluoroquinolone antibiotics (and thus their spectrum of activity) is dependent in part on their affinity for either DNA gyrase or topoisomerase IV or both. One of the most common mechanisms by which bacteria acquire resistance to fluoroquinolones is by spontaneously occurring mutations in chromosomal genes that alter the target enzymes -- DNA gyrase and topoisomerase IV or both. The frequency with which these spontaneous mutations occurs may be in the range of 10-6. The effect of mutations on the activity of an individual fluoroquinolone will vary depending on the number of mutations, the location of the mutations and which target enzyme is affected. If a mutation occurs (either in the gyrA or gyrB gene) that alters DNA gyrase and results in a reduced affinity of the fluoroquinolone antibiotic for this enzyme, the organism will become resistant.

Macrolides: Mechanisms of Action and Resistance

The macrolides are a group of drugs (typically antibiotics) whose activity stems from the presence of a macrolide ring, a large macrocyclic lactone ring to which one or more deoxy sugars, usually cladinose and desosamine, may be attached. The lactone rings are usually 14-, 15-, or 16-membered. Macrolides belong to the polyketide class of natural products.

Antibacterial

Macrolides are protein synthesis inhibitors. The mechanism of action of macrolides is inhibition of bacterial protein biosynthesis, and they are thought to do this by preventing peptidyltransferase from adding the peptidyl attached to tRNA to the next amino acid (similarly to chloramphenicol) as well as inhibiting ribosomal translocation. Another potential mechanism is premature dissociation of the peptidyl-tRNA from the ribosome.

Macrolide antibiotics do so by binding reversibly to the P site on the subunit 50S of the bacterial ribosome. This action is considered to be bacteriostatic. Macrolides tend to accumulate within leukocytes, and are, therefore, transported into the site of infection.

Diffuse panbronchiolitis

The macrolide antibiotics erythromycin, clarithromycin, and roxithromycin have proven to be an effective long-term treatment for the idiopathic, Asian-prevalent lung disease diffuse panbronchiolitis (DPB). The successful results of macrolides in DPB stems from controlling symptoms through immunomodulation (adjusting the immune response), with the added benefit of low-dose requirements.

With macrolide therapy in DPB, great reduction in bronchiolar inflammation and damage is achieved through suppression of not only neutrophil granulocyte proliferation but also lymphocyte activity and obstructive secretions in airways.[6] The antimicrobial and antibiotic effects of macrolides, however, are not believed to be involved in their beneficial effects toward treating DPB. This is evident, as the treatment dosage is much too low to fight infection, and in DPB cases with the occurrence of the macrolide-resistant bacterium Pseudomonas aeruginosa, macrolide therapy still produces substantial anti-inflammatory results

Resistance

The primary means of bacterial resistance to macrolides occurs by post-transcriptional methylation of the 23S bacterial ribosomal RNA. This acquired resistance can be either plasmid-mediated or chromosomal, i.e., through mutation, and results in cross-resistance to macrolides, lincosamides, and streptogramins (an MLS-resistant phenotype).

Two other types of acquired resistance rarely seen include the production of drug-inactivating enzymes (esterases or kinases), as well as the production of active ATP-dependent efflux proteins that transport the drug outside of the cell.

Azithromycin has been used to treat strep throat (Group A streptococcal (GAS) infection caused by Streptococcus pyogenes) in penicillin-sensitive patients, however macrolide-resistant strains of GAS are not uncommon. Cephalosporin is another option for these patients.

Effux pumps

Active efflux is a mechanism responsible for extrusion of toxic substances and antibiotics outside the cell; this is considered to be a vital part of xenobiotic metabolism. This mechanism is important in medicine as it can contribute to bacterial antibiotic resistance.

Efflux systems function via an energy-dependent mechanism (Active transport) to pump out unwanted toxic substances through specific efflux pumps. Some efflux systems are drug-specific, whereas others may accommodate multiple drugs, and thus contribute to bacterial multidrug resistance (MDR).

Bacterial efflux pumps

Efflux pumps are proteinaceous transporters localized in the cytoplasmic membrane of all kinds of cells. They are active transporters, meaning that they require a source of chemical energy to perform their function. Some are primary active transporters utilizing Adenosine triphosphate hydrolysis as a source of energy, whereas others are secondary active transporters (uniporters, symporters, or antiporters) in which transport is coupled to an electrochemical potential difference created by pumping out hydrogen or sodium ions outside the cell.

Bacterial efflux transporters are classified into five major superfamilies, based on the amino acid sequence and the energy source used to export their substrates:

1. The major facilitator superfamily (MFS)
2. The ATP-binding cassette superfamily (ABC)
3. The small multidrug resistance family (SMR)
4. The resistance-nodulation-cell division superfamily (RND)
5. The Multi antimicrobial extrusion protein family (MATE).

Of these, only the ABC superfamily are primary transporters, the rest being secondary transporters utilizing proton or sodium gradient as a source of energy. Whereas MFS dominates in Gram positive bacteria , the RND family is unique to Gram-negatives.

Function

Although antibiotics are the most clinically important substrates of efflux systems, it is probable that most efflux pumps have other natural physiological functions. Examples include:

* The E. coli AcrAB efflux system, which has a physiologic role of pumping out bile acids and fatty acids to lower their toxicity.
* The MFS family Ptr pump in Streptomyces pristinaespiralis appears to be an autoimmunity pump for this organism when it turns on production of pristinamycins I and II.
* The AcrAB–TolC system in E.coli is suspected to have a role in the transport of the calcium-channel components in the E. coli membrane.
* The MtrCDE system plays a protective role by providing resistance to faecal lipids in rectal isolates of Neisseria gonorrhoeae.
* The AcrAB efflux system of Erwinia amylovora is important for this organism's virulence, plant (host) colonization, and resistance to plant toxins.
* The MexXY component of the MexXY-OprM multidrug efflux system of P. aeruginosa is inducible by antibiotics that target ribosomes via the PA5471 gene product[1].

The ability of efflux systems to recognize a large number of compounds other than their natural substrates is probably because substrate recognition is based on physicochemical properties, such as hydrophobicity, aromaticity and ionizable character rather than on defined chemical properties, as in classical enzyme-substrate or ligand-receptor recognition. Because most antibiotics are amphiphilic molecules - possessing both hydrophilic and hydrophobic characters - they are easily recognized by many efflux pumps.

Bleomycin Anti cancer agent

Bleomycin is a glycopeptide antibiotic produced by the bacterium Streptomyces verticillus. Bleomycin refers to a family of structurally related compounds. When used as an anti-cancer agent, the chemotherapeutical forms are primarily bleomycin A2 and B2. Bleomycin A2 .The drug is used in the treatment of Hodgkin lymphoma (as a component of the ABVD regimen), squamous cell carcinomas, and testicular cancer, as well as in the treatment of pleurodesis and plantar warts.

Bleomycin was first discovered in 1966 when the Japanese scientist Hamao Umezawa found anti-cancer activity while screening culture filtrates of S. verticullus. Umezawa published his discovery in 1966. The drug was launched in Japan by Nippon Kayaku in 1969. In the US bleomycin gained Food and Drug Administration (FDA) approval in July 1973. It was initially marketed in the US by the Bristol-Myers Squibb precursor Bristol Laboratories under the brand name Blenoxane.

Mechanism of action

Bleomycin acts by induction of DNA strand breaks. Some studies suggest that bleomycin also inhibits incorporation of thymidine into DNA strands. DNA cleavage by bleomycin depends on oxygen and metal ions, at least in vitro. It is believed that bleomycin chelates metal ions (primarily iron) producing a pseudoenzyme that reacts with oxygen to produce superoxide and hydroxide free radicals that cleave DNA. In addition, these complexes also mediate lipid peroxidation and oxidation of other cellular molecules.

Bleomycin. . In Wikipedia, The Free Encyclopedia. from http://en.wikipedia.org/w/index.php?title=Bleomycin&oldid=270981622

Meropenem Animation

Meropenem is an ultra-broad spectrum injectable antibiotic used to treat a wide variety of infections, including meningitis and pneumonia. It is a beta-lactam and belongs to the subgroup of carbapenem, similar to imipenem and ertapenem. Meropenem was originally developed by Sumitomo Pharmaceuticals. It is marketed outside Japan by AstraZeneca with the brand names Merrem and Meronem. Other brand names include Mepem (Taiwan) Meropen (Japan, Korea) and Neopenem (India) . It gained FDA approval in July 1996. It penetrates well into many tissues and body fluids including the cerebrospinal fluid, bile, heart valves, lung, and peritoneal fluid.

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Once Meropenam reaches Penicillin Binding Protein (PBPs) located at cytoplasm membrane it binds to them and interfere with their ability to create and modify peptidoglycan layer In PSEUDOMONAS AERUNGINOSA, Meropenam has a high affinity to the PBPII and PBPIII, this affinity is be lived to associate with high anti-pseudomonas activity has the result the cell wall weakens, osmotic pressure builds and cell ruptures and dies.

As a defense mechanism, bacteria develops resistance to antibiotics, the most common form of resistance to beta-lactam antibiotics is production of beta lactimases, enzymes that inactivate this antibiotics by breaking up the beta lactin rings.Merapenam retains high activity against PSEUDOMONAS AERUNGINOSA despite this organism inherent resistance to may drug classes. Studies have shown that Meropenam is not easily degrade by beta lactimases in the periplasmic space of Gram-negative Bacteria as other antibiotics. Merapenam has displayed significant stability to beta lactimases of most categories including penicillinases. Merapenam stability is believed to be link to 6alpha hydroxyl ethyl side chain on its chemical structure.