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“Wolf’s liver taken in thin wine, the lard of a sow that has been fed upon grass, or the flesh of a she-ass taken in broth.” –Pliny the Elder

Ring of Inhibition

We’ve come a long way in some ways, at least since “flesh of a she-ass” was considered a specific remedy from disease.  Though, did you know that in early Greece moldy bread was a remedy against infection of wounds?  The mold was presumably a kind of penicillium that produced chemicals inhibiting bacterial growth.  On that fine day in 1928, Alexander Fleming had no idea he would discover something that would change medicine forever.  After coming back from vacation and sifting through the piles of clutter in his office he noticed a culture of staphylococcus in which a blue mold was growing.  The mold had pushed back the staph in a characteristic ring of inhibition.  As the story goes, he hypothesized that the mold must be producing something that retarded bacterial growth.  Penicillium chrysogenum produces the chemical backbone used to produce penicillin, an antibiotic which caused a great leap forward in western medicine.  The effect has been so great that the old killers of yesteryear, the terrible diseases that for ages struck fear in the hearts of man are no longer thought of in the same grave tone and are considered more of a nuisance to public health, a mere trifle in consideration of the far greater concerns of the general populace.  Perhaps the greatest triumph is the reaction of surprise that “people still get that?!”

The back bone of penicillin antibiotics, 6-aminopenicillanic acid, was originally isolated from cultures of Fleming’s Penicillium

Various functional groups can be added to the amino side chain to produce different drugs.

notatum.  It is a secondary metabolic product, released only when the mold is under environmental stress.  Early manufacturing methods were hampered, because finding just the right conditions for the mold to release the chemical proved difficult, thus production was initially low.  When the first human trial in the U.S. began in 1942, doctors used half of the nation’s total production of penicillin to cure him. The medical value placed upon it began a race to massively increase its production. Researchers from both Britan and the US worked tirelessly to not only elucidate the shape of the molecule, but develop better processes for producing it.  In 1943 a nationwide search was held to find better strains from which the B-lactam skeleton could be obtained.  The lucky winner was provided by a moldy cantaloupe from Illinois, which produced many times the amount of penicillin than Flemming’s original.  This strain exceeded expectations and was quickly put to work manufacturing the raw penicillin components.  At the time, it was so prized that during World War II doctors would collect their patient’s urine to isolate and reuse the excreted portion of penicillin.

Today genetically engineered strains manufacture it in large tanks where conditions such as the pH, temperature and diet are all automatically controlled to elicit maximum output (S).  The growth medium is key to the entire process, where the goal is to put them under nutritional strain.  Growth mediums are usually proprietary information, but they typically contain a source of carbon and nitrogen, which are chosen to make them work harder to gain the nutrition they need to survive.  Penicillium notatum prefers the simple 6 carbon sugar glucose, but can also digest a similar sugar called lactose.  This change in sugars forces them to work harder for the same amount energy.  Looking for an edge to outpace their competitors, they produce chemicals like 6-aminopenicillanic acid that impede bacterial growth.  The incubating contents are constantly aerated to allow for maximum growth throughout the tank.  When the growth phase has completed the contents are filtered and the raw product is extracted, concentrated and purified.  The liquid can then be altered chemically and turned into a number of antibiotics by adding functional groups to the base skeleton.

Upon discovery of the antibiotic nature of this compound, researchers put the skeleton through many thousands of reactions in order to find compounds with similar properties.  There are many kinds of penicillin, which differ by the addition of unique functional group.  For example, adding phenyl acetyl chloride to the purified preparation produces benzyl penicillin, or penicillin G, as it is more commonly known.  These new additions can change the properties of the original compound, endowing it with activity against a wider range of bacteria or a specific subset of them.  Penicillin G, for example, is strongest against Gram positive organisms, like staphylococci and streptococci, and gram negative cocci, like N. gonorrhoeae, but has low activity against bacteria that produce enzymes that inactivate the compound, called B-lactamases.  With N. gonorrhoeae, however, there has been increasing resistance of this kind reported and penicillin G is no longer considered the drug of choice to treat them.  According to William Strohl, “Over twenty percent of current isolates of N. gonorrhoeae are resistant to penicillin, tetracycline, cefoxitin, and/or spectinomycin.” (S)

B Lactams usually block the enzyme that ties these pieces together.

With such a wide range of activities against so many organisms one might wonder how they work.  Like all poisons, penicillins inhibit critical life processes in the species attacked. In this case, they inhibit bacterial growth by stopping the repair and replacement of their cell walls.  In affected strains, the cell walls are composed of long chains of six carbon sugars with amino acid side chains that weave the chains together like a net.  The long chains are made by polymerizing N-acetylglucosamine and N-acetlymuramic acid sugars into alternating units.  The side chains are made of repeating amino acid units, which differ per species, but for susceptible species they end with the combination D-alanyl-D-alanine amino acids.  B-Lactam antibiotics are natural analogues of these amino acids, which is to say they can fit into some of the same places as the natural bacterial amino acid piece can.  The antibiotics attach themselves to a special protein (the aptly named ‘penicillin binding protein’) that transports and tethers the D-alanyl-D-alanine amino acids to other chains in the cell wall, blocking its activity(S).  With these proteins blocked the cell wall can’t be replaced or repaired. Lysis results when the cell wall is unable to withstand the osmotic pressure upon it.

It is important to point out that these antibiotics are most active when bacteria are growing and synthesizing cell walls, which is fine for fast growing colonies, like the streptococci.  However mycobacteria, which are famous for causing tuberculosis and leprosy, have a dormant states where they don’t actively synthesize new cell wall components as well as impregnate them with certain lipids that impede the entry of penicillins.  In this context, an attack of this kind would be useless or at the very most a stop gap measure.  Since the colony isn’t completely killed off during treatment, this situation is favorable for the development of antibiotic resistances and the selection of hardy strains of the bacteria.  In a clinical setting these factors complicate issues, with treatments for tuberculosis taking upwards of a year or more to complete.

In a colony of 10^6 bacteria, the probability is that at least one will have resistance to a given antibiotic.  A typical mycobacterial colony will have something around 10^8 members, making the selection of strong, antibiotic resistant strains almost a certainty.  This problem is further compounded by a form of bacterial communication called horizontal gene transfer or plasmid exchange.  This is where a circular strand of DNA, typically encoding a gene producing a resistance, is absorbed by other bacteria allowing the technical means of resistance to spread. This is how bacteria such as the dreaded MRSA have developed and why infections from them are most common in hospitals. In the case of tuberculosis, multi-drug therapies utilizing non-B-lactam antibiotic combinations, like isonaizid-rifampin, can cure 95-98% of cases (S).  The probability that one will have a resistance against both antibiotics is very low and using two will usually kill the bacteria that have a selected immunity to one antibiotic alone.  The other 2-3% typically require specialized treatment and top of the line antibiotics, like moxifloxacin or rifabutin.  However, moxifloxacin resistant strains have been recently identified as a cause for concern (S).

Bacterial resistances usually present via four different routes: Reduced membrane permeability to the drug, production of an enzyme that deactivates the antibiotic (a B-lactamase, for example), a mutation in the PBP site where the antibiotic binds and finally, the creation of a means for drug removal (S).  With penicillins, B-Lactamase production is the most common form of resistance, some of which are highly specific to only one kind of drug (S).  B-lactamases attack the core structure of the penicillin molecule, breaking a critical carbon-nitrogen bond.  Mutations in penicillin binding proteins alter the shape of the binding site, making it harder for the B-lactam structure to fit.  In theory mutations could be great enough to block the penicillin from binding completely.  In practice, however, mutations that cause a PBP to be blocked entirely are often less useful functionally, because these will also block the D-alanyl-D-alanine amino acids they were originally intended for.  Small mutations are more useful to inhibit rather than block the penicillin and keep them from reaching a concentration necessary to inhibit bacterial growth. (S)  Gram negative bacteria usually develop resistances to antibiotic entry by changing the shape of receptors that transport the antibiotic across its membrane.  If the drug can’t pass through the membrane, it can’t attack the cell wall manufacturing machinery.  Another means that changes the membrane permeability is the creation of what is called an efflux pump.  These work to transport the antibiotics back across the membrane after entry.  These are of varied efficiencies, but always work to increase the amount of antibiotic required for efficacy.  Both mutation and changes in permeability are often paired with a B-Lactamase enzyme, making an effective, multi-front strategy for resistance.

Even though we have produced effective weapons against pathogenic bacteria, they have begun to fight back.  They have been largely successful in this regard, calling into question how long the usefulness of these drugs can be maintained.  This has spurred the demand for newer drugs to combat the potential ‘super-bugs’ on the horizon and limited use policies to maintain drug effectiveness in the future.  All in all, penicillin is generally considered non-toxic.  The worst adverse reactions are due to hypersensitivities.  Allergic reactions occur, however, this is a rarity occurring in less than 1% of people and is usually not life threatening (a rash, fever, etc).  Even less have a serious reaction, like anaphylactic shock (0.05% of patients).  In a worst-case scenario, though, desensitization to the drug can be used to effectively treat a given disease.

One thing is for sure is that we will continue to look in awe at the complexity life has to offer.  We hope that new attempts to cultivate and extract new antibiotics will be successful in the future.

{sources available upon request}

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Enterobactin

A little over 40 years ago it was discovered that bacteria utilize a class of metal chelators called siderophores to capture iron from their environments.  Iron is an important element for the growth and development of most kinds of bacteria.  It is used as a critical element in electron transport chains for energy production, the removal of dangerous reactive oxygen species and the functional element in the reactive sites of many important enzymes.  The finding was critically important to the field of medicine, as the iron scavenging activities of pathogenic bacteria are most often detrimental to the host.  Even though humans and other animals have developed sophisticated systems to sequester iron and make the body inhospitable to these bacteria, likewise they have developed a variety of means of out-competing their hosts for the valuable element.  Neisseria gonorrheae and N. meningitidis, for example, have evolved the ability to directly capture iron containing lactoferrin proteins from their human hosts, a testament to our continued co-evolution (1).  Still others have relied upon producing their own iron scavenging siderophores to fish out this valuable metal from fluid and protein sources.  Escherichia coli , for example, are known for producing enterobactin and have one of the most effective iron scavenging systems known, which at biological pH easily out competes endogenous defenses for iron.

Recently, it has been noted that these compounds are of critical importance to bacterial growth, especially in the ultra-low iron concentrations of bodily tissues.  Rather than tighten their belts in the toughest of times, some kinds of pathogenic bacteria turn on

'Like dissolves like': different kinds of Mycobactin are used in different mediums. The long aliphatic chain endows fat solubility to the molecule, allowing it to shuttle through the waxy mycobacterial membrane easily.

these scavenging systems to forcibly acquire the iron needed for growth.  Mycobacterium tuberculosis produces several siderophores, called mycobactins, that are medium specific in their activities.  The two differ by a water soluble or fat soluble group attached to the siderophore skeleton.  Water-soluble mycobactin T is released into aqueous mediums, whereas fat soluble mycobactin T is designed to take pirated iron across the waxy mycobacterial cell membrane (2).  When iron is in good supply, however, production of these is unnecessary and is down-regulated to conserve valuable energy and resources.  Below the level of production of these siderophores, however, lies a regulated system of control.

Generally siderophore production is controlled by gram negative bacteria’s “Fur” and gram positive’s “DtxR” systems, which have  transcription elements that are bound by iron.  Thus, when intracellular iron concentrations fall below a threshold (approximately 10^-6 M for many pathogenic bacteria) the iron lynchpin holding back the transcription element is gone, activating the siderophore production system.  In 1999, researchers at the University of Queensland showed that Mycobacteria require the production of siderophores for growth in human tissues and macrophages (2).  They used genetic engineering techniques to remove the instructions for the enzyme MbtB, which is a critical component of mycobactin synthesis.  MtbB effects the final step in mycobactin synthesis, the coupling of a salicylic acid group to the siderophore’s finished structure.  After creating an MtbB knockout strain, its ability to produce siderophores was tested by an assay that measured the absorbance of the growth medium.  They used a colored dye compound that has a known affinity for iron in solution.  If a bacteria were to produce something that has a higher affinity for iron than the dye, this would be measured as a drop in absorbance, as the siderophore would compete with the dye for the iron.  They found that after comparing these bacteria against wild type strains that they were unable to produce siderophores and the deficient strains experienced retarded growth both in plate medium and in live macrophages (in vivo vs. in vitro tests).

The understanding of how important these chemicals are for bacterial growth has led to renewed attempts to develop antibiotics against them.  In the late 80 and early 90’s the ‘trojan horse’ tactic of using siderophore-antibiotic conjugates and piggybacking them into a cell began to bear fruit with successes being discovered in a wide variety human diseases (3)(4)(5).  One group of scientists developed conjugates using the carbacephem antibiotic, loracarbef.  They found that the mixed catecholate-hydroximate siderophore they used gave the conjugate compound 2,000 times more potency than the parent drug alone (6).  Though resistances to this tactic still developed, these are thought to leave the bacteria at a disadvantage, because the mutations hinder the siderophore uptake system rather than attack the antibiotics themselves.  This limits access to a vital nutrient and leaves the mutants more susceptible to iron starvation and hindered growth.

Though the trojan horse tactic has yielded positive results, there are opportunities for even greater exploitation.  Techniques involving small molecules that attack that actual production of siderophores could provide another avenue to beneficial therapy for these diseases.  These drugs, much like statins that block the enzyme HMG-coA, would block a critical piece of the siderophore production pathway.  In the particular case of mycobactins, the final step in biosynthesis is the attachment of a salicylate group to the mycobactin skeleton.  Researchers at Cornell published results in 2005 showing that inhibitors of the enzymes that accomplish this task are potent inhibitors of M. tuberculosis and Yersinia pestis (7).  They synthesized a compound, SAL-AMS, that closely resembled a reaction intermediate and measured its effect upon the growth of bacterial cultures in mediums of low iron concentration.  They found that it successfully inhibited the enzyme and drastically reduced bacterial growth in the cultures.  It was shown to have an IC50(*) of 2.2 ± 0.3 μM for M. tuberculosis and about 51.2 ± 4.7 μM for Y. pestis in an iron limited medium.  Though in mediums with high concentration of iron the chemical was ineffective against Y. pestis, but it was found that the chemical might have unknown inhibitory properties against M. tuberculosis, as:

“Salicyl-AMS (tested at up to 8 X IC 50) was not active against Y. pestis in iron-supplemented medium, in which siderophore production is not required for growth.  Under these conditions, salicyl-AMS (tested at up to 180 x IC50) did inhibit M. tuberculosis growth, albeit with an 18-fold increase in IC50 (39.9 ± 7.6 μM).  This suggests that, in addition to blocking siderophore biosynthesis, salicyl-AMS may also inhibit M. tuberculosis growth by other mechanisms.”

Furthermore, researchers at the university of Minnesota developed similar nucleoside inhibitors of MbtA, one of which ” rivals the first-line antitubercular isoniazid” in activity against the bacteria (8).  This tactic follows the same reasoning as SAL-AMP above, as the nucleoside inhibitors attack the same pathway to inhibit siderophore end production.  The research was centered around making logical functional modifications to known structures of inhibitors and choosing the most effective ones for further testing.

Comparison of the pathway intermediate, the Cornell inhibitor and one of the Minnesota nucleoside inhibitors.

These findings are coming just in the nick of time it seems, as certain drug resistant strains of M. tuberculosis have become big news recently.  Extensively drug resistant tuberculosis is a kind of tuberculosis that is resistant to at least two of the top line drugs used to normally treat it (typically isoniazid and/or rifampicin) and a member of the quinolone antibiotics (ciprofloxacin).  Tuberculosis is generally a challenge to treat in the first place, with treatments typically taking up to a year or more to complete.  The loss of the first line drugs and reliance upon second line increases the risk of side effects and patient noncompliance to the already long course of therapy.    This can further complicate the issue, as it could lead to the obsolescence of the few active  drugs used to treat the disease, because resistances to one drug are usually useful against the whole family of drugs.

β-lactamases typically attack the carbonyl in the β-lactam structure, destroying the ring. Nafcillin has a large group that hinders these enzymes from getting too close.

An example of this can be seen in bacteria that produce β-lactamases, as these strains are often cross-resistant to all unprotected β-lactam antibiotics.  Some  penicillins have been designed with bulky groups attached to the skeleton in an attempt to hinder these enzymes.  Nafcillin, with its large 2-ethoxy-1-naphthoyl group, is very effective at blocking these enzymes for the most part.  However, even this tactic has its limits as methicillin resistant Staphylococcus aureus (MRSA) and oxacillin resistant Staphylococcus aureus (ORSA), both have developed resistances against these drugs such that, “From 1999 through 2005, the estimated number of S. aureus–related hospitalizations increased 62%, from 294,570 to 477,927 (9),” and “MRSA accounts for an estimated 12% of all nosocomial bacteremias, 28% of surgical wound infections, and 21% of nosocomial skin infections.  Infections secondary to MRSA result in excess costs of approximately $4,000 per patient per hospitalization compared with patients infected with methicillin-susceptible S aureus (MSSA)”(10)(11).

Since the discovery of penicillin, Alexander Fleming hypothesized that bacteria would develop resistances to the antibiotics used to treat them.  Now, more than ever, the development of new antibiotics must be pursued.  Human defenses have always provoked a counter-response from our pathogens, the most successful tactic selected out.  Much like the pathogens that infect us, we must ‘evolve’ a newer understanding of bacterial biology, which will offer us a foothold to better, more effective treatments.

——

(1) Genetics and Molecular Biology of Siderophore-Mediated Iron Transport in Bacteria

JORGE H. CROSA

(2) The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages

James J. De Voss, Kerry Rutter, Benjamin G. Schroeder, Hua Su, YaQi Zhu, and Clifton E. Barry III

(3) Design, Synthesis, and Study of a Mycobactin−Artemisinin Conjugate That Has Selective and Potent Activity against Tuberculosis and Malaria

Marvin J. Miller, Andrew J. Walz, Helen Zhu, Chunrui Wu,Garrett Moraski, Ute MÖllmann, Esther M. Tristani, Alvin L. Crumbliss, Michael T. Ferdig, Lisa Checkley, Rachel L. Edwards, and Helena I. Boshoff

(4) Species Selectivity of New Siderophore-Drug Conjugates That Use Specific Iron Uptake for Entry into Bacteria

M. S. DIARRA, M. C. LAVOIE, M. JACQUES, I. DARWISH, E. K. DOLENCE, J. A. DOLENCE, A. GHOSH, M. GHOSH, M. J. MILLER,    AND F. MALOUIN

(5) Siderophore-Based Iron Acquisition and Pathogen Control

Miethke M, Marahiel MA.

(6) Iron Transport-Mediated Antibacterial Activity of and Development of Resistance to Hydroxamate and Catechol Siderophore-   Carbacephalosporin Conjugates

ALBERT A. MINNICK, JULIA A. McKEE, E. KURT DOLENCE, AND MARVIN J. MILLER

(7) Small-molecule inhibition of siderophore biosynthesis in Mycobacterium tuberculosis and Yersinia pestis

Julian A Ferreras, Jae-Sang Ryu, Federico Di Lello, Derek S Tan & Luis E N Quadri

(8) Antitubercular Nucleosides That Inhibit Siderophore Biosynthesis: SAR of the Glycosyl Domain

Ravindranadh V. Somu, Daniel J. Wilson, Eric M. Bennett, Helena I. Boshoff, Laura Celia, Brian J. Beck, Clifton E. Barry, III, and Courtney C. Aldrich

(9) Hospitalizations and Deaths Caused by Methicillin-Resistant Staphylococcus aureus, United States, 1999–2005

Eili Klein, David L. Smith, and Ramanan Laxminarayan

(10) Baquero F. Gram-positive resistance: Challenge for the development of new antibiotics. J Antimicrob Chemother. 1997;39(suppl A):1–6.

(11) Kopp BJ, Nix DE, Armstrong EP. Clinical and economic analysis of methicillin-susceptible and -resistant Staphylococcus aureus infections. Ann Pharmacother. 2004;38:1377–1382.

(*) The IC 50 is the half maximum inhibitory concentration of antibiotics, a typical measure of effectiveness of the drug.

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Back in the early days of photosynthesis, the oxygen released into the atmosphere created an oxidative environment. This converted more soluble forms of iron into the familiar orange-red, insoluble Fe+3 we see on a daily basis as rust. Iron is needed by microbes to support all sorts of biological processes, like respiration, but in the Fe+3 state it is hard to acquire and even harder to manipulate in a useful manner. Its use in cytochrome proteins of the electron transports system should underline its importance in this role. Further, in mammals the element is tightly bound to proteins such as the famous hemoglobin in red blood cells and transferrin, a mammal iron scavenger. This sequestration of iron creates a bactericidal environment inside the body and forces the invading bacteria to compete for sources of iron to fuel their biological processes.

Since it is critical to the growth and function of bacteria, especially pathogenic bacteria, it is not surprising that they have developed a variety of means to acquire this element. Some of their negative effects on a host organism directly result from this scavenging activity. Some produce proteins called hemolysins to puncture cells and release their contents, and/or produce compounds called siderophores, which are iron scavenging compounds used to leach iron and iron containing proteins from the environment.

These compounds are ubiquitous throughout the microbiological world and are usually produced under conditions of low iron concentration, like those of sterile sites in the body. While finding bacteria in cultures from skin and naso-pharyngeal swabs is not necessarily an indication of disease, finding bacteria in cultures taken from cerebral spinal fluid and blood (etc) is considered an important point in diagnosis. The skin functions as a barrier to infectious microbes, but it can be breached by pathogenic critters. Essentially, for entry into the body a microbe (or chemical) will have to cross a tissue called an epithelium. Hemolysins and cellular matrix degrading proteins are used to damage this layer and gain entry into the body and access to essential nutrients like iron. Siderophores are used to scavenge the iron from the contents of dead cells.

Siderophores vary widely in structure per species, but one thing they have in common is their preference for Fe+3. Fe+3 ions are unlike the typical organic ‘binds 4 times’ carbon atoms. Iron has a variety of oxidation states available to it and the Fe +3 state has six coordination sites arranged in the shape of an octahedron. The siderophore binding site usually accommodates this by being shaped similarly and employing atoms that also have a strong affinity for iron, specifically oxygen. Often, they employ functional groups such as catecholates and hydroxamates that can bind strongly to ions in general. These are usually arranged in such a way as to virtually guarantee that Fe+3 will be the only ion taken in by the protein. Enterobactin is a prime catecholate example of this set up, with is produced generally by enteric bacteria like Escherichia coli. Ferrichrome is another well known example of a hydroxamate compound. The binding affinity for these kind of +3 ions is very high (Ga+3 binding is also very strong), often selectively much more than +2 ions like Al+2. The fact that there is a big difference between the binding efficiency of these two oxidation states affords an efficient means of releasing the iron once it has been captured. Once the bacteria have the iron, they can reduce it to a more soluble and useful Fe+2 state.

Most bacteria have specialized receptors to accommodate these proteins, but this fact also leaves them open to attack. Bacteriophages, bacteriocins and antibiotics have been known to utilize the ferrichome transport receptors for entry into the cell. Bacteriocins are a large class of bacterially produced antibiotics used to compete with other similar strains of bacteria. Some of these have found use in medicine and agriculture to inhibit some pathogenic strains of bacteria in humans and livestock. More to the point, a new thought in antibiotic production is using these siderophore functional groups to cause their uptake by bacterial iron transport systems. The high specificity for these groups and the difficulty in changing the receptor’s structure by mutation (mutation may cause less specificity and inhibit the uptake of a vital resource) make this an interesting tactic.

By attaching a sidereophore moiety to the skeleton of an ordinary antibiotic, its potency can be increased and the effective dose lowered. In the case of attachments to the cephalosporin class of antibiotics, this effect has been studied and proven since the late 1980’s. Studies have shown that experimental catecholate cephalosporins have lower minimum inhibitory concentrations, than comparable antibiotics (frontline ceftriaxone and/or ceftazidime) making the effective dose lower than ordinary antibiotics.

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