Discussing the Nature and Future of Antibiotics - Part 1

Antibiotics are chemical substances that either inhibit the growth of or terminate bacteria. Typically antibiotics are restricted to bacteria and have no significant effect against viruses or other pathogenic agents due to their points of action. The verbal origins of the word antibiotic come from the Greek anti (against) and bios (life). Despite the existence of the immune system, before the development of antibiotic treatment protocols and their widespread use many illnesses like pneumonia, tuberculosis and typhoid carried very high fatality rates. Now when discounting for bacterial resistance fatality rates from all manner of infection are much lower.

Most of the early work concerning antibiotics occurred between the years 1928 and 1942 with the initial work of Sir Alexander Fleming in 1928 who accidentally empirically discovered the anti-bacterial properties of the mold, Penicillium and Gerhard Domagk who discovered the first class of antibacterial agent, sulfonamides, in 1935. In 1939 Rene Dubos discovered the first naturally derived antibiotic, tyrothricin, but after testing was deemed too toxic for human administration. The substance utilized by the mold to achieve such properties was isolated and unsurprisingly named penicillin by Ernst Chain and Howard Florey in 1942. By the early 1940s both sulfonamides and penicillin began clinical use in treatment of diseases. Later penicillin was first chemically synthesized in 1957 by John C. Sheehan, which generated the basic information required to begin synthesis of synthetic penicillin derived antibiotics to combat resistance.

Antibiotics have two modes of function: prevention of bacterial growth (bacteriostatic) and bacterial death (bactericidal). In normal immune systems typically either function is sufficient for recovery as the patient’s immune system should be able to neutralize the bacteria after antibiotic application. However, in those individuals who have weaker immune systems, elderly, very young children or other immunocompromised, bacteriostatic antibiotics may not be enough to fully treat the illness. The bactericidal behavior of penicillin is largely why it was the favored antibiotic over sulfonamides before bacterial resistance started to emerge.

Antibiotics have a wide range of functional pathways to influence bacterial growth: 1) destroy cell wall either through the production of pores or negation of synthesis elements; 2) neutralize aspects of protein synthesis including blocking ribosome production or their binding to appropriate targets; 3) neutralize DNA synthesis and other nucleic acid metabolism functions.1-4 When identifying a new antibiotic agent testing is important to determine the functionality of the antibiotic, how long it works and how large of a dose is required to receive an effective treatment response. Dosage is important because a large enough dose of antibiotic administered against a non-resistant target will kill it, but will also generate significantly detrimental side effects as well.

One of the major targets for antibiotics currently utilized in treatment is the bacterial ribosome largely because of its significant structural differences relative to its mammalian cousin. Bacterial ribosomes are 70S with a 30S and 50S subunit that are comprised from three types of rRNA (5S, 16S and 23S) versus the 80S mammalian ribosome.2,5 This difference makes antibiotics that target ribosomes attractive candidates as the probability of antibiotic interaction against mammalian (friendly) ribosomes leading to unpleasant side effects is low. Antibiotics that target DNA synthesis can also be attractive candidates because of the ability to target molecules that are not utilized in mammalian cells like bacterial DNA girase (topoisomerase II),6 which packs and unpacks supercoiled bacterial DNA, or bacterial DNA-dependent RNA polymerase.7 Other antibiotics neutralize bacteria through indirect means like targeting unique secondary products that are required for DNA like the metabolism of tetrahydrofolic acid. Tetrahydrofolic acid is essential for the synthesis of purines, pyrimidines and some amino acids. Anti-metabolites interfere with tetrahydrofolic acid synthesis and, therefore, inhibit DNA synthesis.

There are two classifications for antibiotics: narrow-spectrum that only work on a small number of specific bacteria and broad-spectrum that work against a large number of bacteria. Not surprisingly though because of its target breadth the use of broad-spectrum antibiotics has a higher probability of developing antibiotic resistant bacteria. Therefore it is standard operating procedure for broad-spectrum antibiotics to only be assigned when the pathogen is unidentified or if unresponsive to any narrow-spectrum antibiotics. For instance gram negative bacteria are typically treated with broad-spectrum antibiotics due to the structural differences in the cellular wall and internal reproduction machinery, which make more narrow-spectrum antibiotics, which are designed to treat gram positive bacteria, ineffective.

Antibiotics are also separated into three classifications derived from their application: surface, oral or intravenous. Surface applications are placed on the skin, in the eyes or mucous membrane in the nose and are typically limited to the local area around where the antibiotic is applied. Oral applications are pills, tablets and gel-caps that are swallowed breaking down in the small intestine and are later absorbed by the bloodstream. Intravenous application is the most powerful application because there is little residence or lag time between application and absorption into the bloodstream. However, intravenous application typically only occurs at a hospital and is largely reserved for critical or uniquely specific conditions.

Of the three classifications oral application is one of the principle elements responsible for the development of bacterial resistance because despite warnings a number of patients continue to prematurely cease oral treatments due to a disappearance of illness related symptoms. Basically patients take some of the assigned dosage start to feel better and fail to finish the remainder of the dose due to a belief of non-necessity. Unfortunately not completing the dosage increases the probability of surviving bacteria becoming resistant.

Although antibiotics are normally used to treat illness they are also prescribed in certain situations to reduce the potential for infection and illness. The most common scenario for this preventative strategy is antibiotic treatment before major surgery to reduce probabilities of current operative and post-operative infection. Also combination therapy is popular where multiple drugs are administered together where one mechanism aids the effectiveness of the other in a synergistic relationship. An example of such a relationship is penicillin weakening or destroying cell walls allowing aminoglycoside entry into the cell. Typically synergistic activity is not an effective treatment strategy outside of circumventing non-pathway resistant mutations because normally a single antibiotic and the innate immune system can neutralize an illness. Also while effective combination therapy failure increases resistant probabilities for multiple drugs instead of just one.

Unfortunately the second most common pathway for bacterial resistance has developed from the widespread use of antibiotics in animal and milk production. In short farmers and corporate entities apply large amounts of antibiotics to healthy dairy cows and other livestock in an attempt to reduce their probability of contracting illness and increase their weight. The weight increases come from eliminating the current microbiota (gut bacteria population) through antibiotic treatment and re-establishing a weight-gain favoring microbiota through diet. However, the haphazard application of antibiotics in such a fashion increases the probability that resistant bacteria emerge and because resistance is just a matter of probability frivolous application strategies like the one above have net detrimental outcomes over the long-term.

Although the tools exist to synthetically create antibiotics from a new base core, a vast majority of antibiotics in use are derived from living organisms, mostly molds, fungi and other bacteria. The two major reasons synthetic antibiotics have not become prominent are safety and functionality concerns along with the financial cost associated with creating antibiotics versus the “lack” of a market for pharmaceutical profit. One of the most common and reliable ways of producing an antibiotic is biosynthesis where the specific organisms themselves manufacture the antibiotic under optimized growing and conditions with additional elements/stressors that will increase the rate and probability for production of the desired elements.

Industrial mass production of antibiotics through biosynthesis is carried out by fermentation where the antibiotics, which are typically secondary metabolites, are collected before cell death. The typical isolation process involves killing the cell, thereby production schemes require large amounts of cell growth to maximize product collection to ensure efficiency and profitability. Collection first involves extraction and then purification into a crystalline product. Organic solvents are used to increase the efficiency of collecting soluble products, but non-soluble products must be removed through additional steps like precipitation, ion exchange and/or adsorption.

The synthesis of synthetic antibiotics typically follows the same methodology. First, an existing antibiotic is selected for modification. The reason behind selecting an existing antibiotic is two-fold: first, there is already empirical certainty that the selected antibiotic has some form of activity against bacteria, thus there is no wasted money developing random chemical structures that have unknown activity profiles. Second, the existing antibiotic is safe enough for human consumption due to widespread use with relatively known side effects.

The second step for synthetic generation is to identify the chemical structure of the active portion of the antibiotic in order to determine what structural modifications could be made to change activation potentials against resistant organisms. Third, the operational pathway of the antibiotic is identified and confirmed. Finally, alterations are made to some part of the structure of the antibiotic to possibly change its response to a given infection. For example all members of the penicillin family have identical rings, but the chemical chain (R group) attached to the ring will be different for different members of the family. So modification of that particular chemical chain is frequently the target of synthetic strategies for penicillin.

After their production these new synthetic derivatives are tested for effectiveness and to ensure retention of safety due to the changes in the chemical formula. In addition to this general methodology new techniques are being used to alter the genetic structure of certain bacteria so the bacteria itself produces a similar, but different antibiotic.

Pharmaceutical companies use computers to data mine and test modifications in the ring to observe chemical compatibility and probability. A standardized testing structure known as 'rational design program' is frequently used now. Such a program focuses on more in-depth analysis of how the favorable agent inhibits by looking at specific targets. In addition to making slight changes in chemical composition to increase antibiotic effectiveness against resistance emergence, another common goal of synthetic antibiotic creation involves increasing the half-life of the drug so that lasts longer in the bloodstream, which will increase the probability for effective treatment.

Identification of side effects and efficiency of treatment is carried out through a screening process that uses a large number of isolates of microorganisms and the secondary products of these organisms are tested in diffusion and growth limitation studies on test organisms. Molecules that show favorable results in both of these areas are then tested for selective toxicities. Afterwards the best candidates are isolated for more rigorous study, which enter the standard three clinical trial phase testing methodology for clinical drugs. However, recently there has been some momentum for changing antibiotic specific testing protocols to speed the application of new antibiotics to the market in effort to better address increasing bacterial resistance.8

Realistically a large amount of natural and synthetic antibiotics have been created at one time or another, but only a select handful have been proven safe and effective. The ideal characteristic of an antibody is a selective bacteria-unique target that negatively influences a critical system required to maintain life. This method of action will lead to the death of the bacteria, but should not interact with any eukaryotic cell targets thus heavily limiting, if not eliminating, any side effects associated with the use of the antibiotic. However, due to the general effectiveness of the immune system antibiotics that lack selective targeting are still useful if administered in a controlled and proper dose. Even if dosage is proper there is valid probability for the development of side effects, thus these changes must also be monitored.

When determining whether or not to treat with antibiotics the first step is to identify what type of organism is responsible for the illness. Under normal circumstances once identification has concluded available treatment options and treatment is rather straightforward. There are typically two elements that complicate treatment strategies. First, if the cause of the illness is viral most treatment options are no longer viable limiting options to a small number of interferons. Second, if the patient is allergic to the primary option of treatment a secondary, more than likely, less effective option will need to be utilized. If the illness is caused by an unidentified pathogen treatment is usually applied using broad-spectrum antibiotics, a strategy commonly called empiric therapy. Overall once a treatment is applied its effectiveness is determined by how well the drug is absorbed into the bloodstream, the diffusion rate of the drug and the half-life of the drug.

Some of the more common classes of antibiotics that have been used in the past or are currently in use are:

Penicillins:

Alexander Fleming discovered the penicillin group from the fungi Pencillium in 1928 (specifically penicillin G was isolated first and later followed by procaine penicillin, benzathine penicillin and penicillin V). Penicillin functions by damaging or destroying bacterial cell walls while the bacterium are in the process of reproduction. The mechanism of action is the inactivation transpeptidase, which is necessary for cross-linking and proper cell wall synthesis. The bacterium accepts penicillin as a substrate and then alkanolates a nucleophilic oxygen of the enzyme, rendering it inactive. Cell wall construction and maintenance ceases leaving multiple holes in the existing cell wall due to continuing natural degradation. This process is also aided by negative feedback where increasing osmotic pressure increases the probability of cytolysis and excess peptidoglycan precursor, due to limited cell wall synthesis, triggers hydrolases and autolysin activation further breaking down the cell wall.

The antibiotic nature of the penicillin is due to the strained beta-lactam ring; when the ring opens, strain is reduced making penicillin more reactive than ordinary amides. Based on its method of action penicillins do not act against organisms lacking cell walls like eukaryotic cells and certain types of bacteria (like most Gram-negative). Due to their ability to facilitate cell wall breakdown, penicillin works well in combination treatments with intracellular acting antibiotics that attack DNA, RNA and/or protein synthesis. Unfortunately due to unsurprising overuse, penicillin has been used for over half a century most bacteria have become resistance on a large-scale.

While the lack of cell walls in eukaryotic cells limits the side effects from these antibiotics, some mild side effects can occur from their use like diarrhea, rashes and hives (which usually indicates an allergy). The most rare and dangerous side effect is an “anaphylactic” allergy, which results in labored breathing due to swelling in the airway born from a severe allergic reaction. Normally allergic reactions eliminate the availability of a given antibiotic, but if the situation demands the use of an antibiotic that will result in an allergic reaction a desensitization strategy is used where the patient receives small doses with a high frequency of administration and slight increases in the overall dosage.

Cephalosporins:

Along with cephamycins, cephalosporins are a class of beta-lactam antibiotic that originally acted against Gram-positive bacteria, but through modern synthetic synthesis newer formulations have increased action effectiveness against Gram-negative bacteria while weakening their action against Gram-positive bacteria. While its method of action is similar to penicillin due to a small structural difference cephalosporins are less susceptible to penicillinases making it more difficult for bacteria to neutralize its bactericidal action. Differing generations of cephalosporins have been created by largely modifying its nucleus to reduce the probability that bacteria develop resistance. Currently, although somewhat disputed, five different generations of cephalosporins have been created with the fourth and fifth generations classified as having broad-spectrum action.

Quinolones:

Quinolones, the largest sub-class being fluoroquinolones, are synthetic broad-spectrum antibiotic that was first developed in 1962 as a distillate byproduct in chloroquine synthesis. Quinolones are typically reserved for secondary application in community-acquired infections due to concerns over peripheral neuropathy along with other strong side effects and increasing bacterium resistance development along with enhancing probability of Clostridium difficile and MRSA infections. However, when used quinolones function occurs through blocking the action of DNA gyrase in Gram-negative bacteria and topoisomerase IV in Gram-positive. For treatment quinolones can stand alone due to their increased probability of cell entry through porins, but in Gram-positive bacteria combination treatment with a beta-lactam antibiotic also increases treatment outcomes. Unfortunately numerous pathogens now demonstrate resistance to quinolones due to overuse in that they were the principle treatment of Gram-negative bacteria from the 60s until the 90s until the development of synthetic broad-spectrum beta lactam antibiotics and continued misuse in the 00s.

Aminoglycosides:

Aminoglycosides are molecules composed of amino-modified sugars and typically can act as antibiotics through two different mechanisms: first the ability to bind irreversibly to the 30S ribosomal subunit in bacteria interfering with successful mRNA interaction reducing the effectiveness of translation (i.e. protein synthesis). There is also some evidence that they also interfere with proofreading and inhibit ribosomal translocation. Second, aminoglycosides are thought to competitively displace magnesium and calcium that link polysaccharides within lipopolysaccharide groups creating transient holes disrupting cell wall permeability.

Aminoglycosides are primarily effective against Gram-negative aerobes and mycobacteria, but while effective antibiotics they are unfortunately rapidly dissolved in the stomach, which eliminates oral consumption, limiting their administration to IVs or injections. Also aminoglycosides have some fairly damaging side effects including kidney and auditory damage. Due to these side effects aminoglycosides are typically used as a last resort antibiotic against unknown or multi-drug resistant pathogens.

Polymyxins:

Polymyxins damages cell walls through interaction between lipopolysaccharide in the outer membrane and its hydrophobic tail in a similar method as detergent/soap. Polymyxin appears to work best in combination treatments with intracellular acting antibiotics for the disruption of the outer membrane allows for easier entry to the cell. Unfortunately similar to aminoglycosides, polymyxins are typically reserved for late stage treatments due to serious neurotoxic side effects.

Tetracyclines:

Tetracyclines (tetracycline, doxycycline) bind to the 16S portion of the 30S ribosomal subunit and block polypeptide chain elongation by preventing the attachment of charged aminacyl-tRNA. They are typically used when treating Gram-positive cocci, chlamydia, mycoplasma, and rickettsia. A secondary mechanism of function involves tetracyclines binding to ions on the membrane and generating additional ionophores disrupting calcium flow into and out from the bacteria. Unfortunately bacteria resistance has developed rapidly against tetracyclines significantly limiting their functionality.

Rifampicin:

Rifampicin binds strongly to DNA-dependent RNA polymerase leading to the inhibition of RNA synthesis. It is largely used in combination treatments against Mycobacterium infections most notably tuberculosis. Due to its mechanism of action rifampicin is also used in secondary combination treatments against more resistant bacteria. Combination treatments are the norm with rifampicin because when used alone bacterial resistance builds quickly.

Macrolides:

Macrolides are molecules with a macrolide ring and a standard construct of a linear molecule that later form a large ring induced by activation of an enzyme on the terminus molecule. These rings are linked through glycoside bonds with amino sugars usually cladinose and desosamine. The linear molecule is constructed similar to that of a protein with small molecules combined on a biological assembly line. Most macrolides function by interfering with bacterial ribosomes by binding reversibly to the P site on the 50S subunit preventing ribosomal initiation complex formation or amino-acyl translocation. In low doses it is typically bacteriostatic, but at higher concentrations it becomes bactericidal, but obviously side effects become more prominent as well. Leukocyte accumulation and interaction aid in transporting macrolides to infection sites increasing efficacy.

Sulfonamides:

Not all sulfonamides are antibacterial, but those that are typically have structures that resemble para-aminobenzoic acid (PABA), which is a precursor in folic acid synthesis. Sulfonamides compete (competitive inhibitors) with PABA to bind to dihydropteroate synthase, an enzyme involved in folate synthesis. Reduction of folate synthesis efficiency makes sulfonamides bacteriostatic. Sulfonamides are typically used in combination treatment, usually erythromycin or trimethoprim, due to recent escalation in resistance. There are few side effects associated with sulfonamide use due to the lack of biosynthesis of folate in humans; folate is instead acquired through diet.

Trimethoprim:

Trimethoprim (TMP) is a synthetic antibiotic. Similar to sulfonamide, trimethoprim selectively inhibits the dihydrofolic acid reductase of bacteria, which converts dihydrofolic acid to tetrahydrofolic acid, a precursor of purines stopping DNA synthesis and replication. TMP is normally bacteriostatic, but can be bactericidal and is commonly used in combination with sulfamethoxazole (SMX). This combination is useful because bacteria that are partly resistant to either TMP or SMX can still be killed by the combination of the two.

Vancomycin:

Vancomycin is a glycopeptide that inhibits cell wall synthesis in Gram-positive bacteria by preventing the synthesis of the long polymers N-acetylmuramic acid and N-acetylglucosamine and preventing polymer cross-linking. Through most of its lifetime vancomycin was viewed as a treatment of last-resort due to its poor oral bioavailability requiring intravenous treatment, which reduced the probability of bacteria resistance. Unfortunately in recent years bacteria resistance has grown exponentially significantly limiting the usefulness of vancomycin.


Resistance has become one of the biggest “rarely talked about” problems in the modern world. A bacterium becomes resistant to an antibiotic when it alters a portion of its genetic structure to neutralize the method of antibiotic action. The genetic change can neutralize antibiotic activity in multiple ways: the target is eliminated entirely, a new defense element is created to eliminate the ability of the antibiotic to target the molecule (deactivating it or removing it) or a change occurs in the pathway of operation eliminating the necessity of the target for growth and survival.1,9 There are typically two major categories that bacteria can alter their genetic structure to create resistance.

Random genetic mutation is the most basic way resistance is acquired. Basically random genetic mutation is the egg in the chicken and the egg question regarding bacterial resistance as no other forms of resistance could occur without the original underlying mutation. Unfortunately these mutations are random and do not require previous exposure to the antibiotic to become resistant. The spontaneous mutation of a susceptibility gene has a frequency of occurrence of anywhere from 10-12 - 10-7 which for some species increases to 10-7 to 10-5 under selective pressure during or after exposure to a specific antibiotic.10 This increased resistance probability is what creates concern by certain parties when discussing frivolous antibiotic administration on healthy organisms.

Another means of mutation that generates resistance is genetic transposition. Transposition is the recombination of genetic material due to transposons which are DNA segments with specific insertion sequences at each end. Using these sequences, transposons are able to migrate or jump between DNA strands in the same bacterium. Sometimes the new sequences that are created due to this movement produce antibiotic resistance.

The second method that leads to resistance is acquisition of a plasmid with the appropriate resistance. Bacteria commonly exchange circular pieces of DNA smaller than their chromosomes otherwise referred to as plasmids. Plasmid acquisition typically occurs in one of three ways: 1) bacterium in close contact with each other freely transfer various plasmids typically referred to as transduction. If the receiving bacterium transfers a plasmid back then the process is referred to as retrotransfer; 2) the unilateral transfer of a plasmid between bacterium during reproduction typically referred to as conjugation; 3) the absorption of a portion or entire chromosome by another bacterium typically referred to as transformation;

Overall there are five major mechanisms in which a bacterium uses these genetic additions or mutations to acquire resistance. 1) Antibiotic Modification - The antibiotic is inactivated typically by the production of a new enzyme that targets the antibiotic changing its structure; 2) Antibiotic Entry Restriction – Modification lowers the probability that the antibiotic is able to enter the cell to target intracellular molecules; this mechanism is more common in Gram-negative bacteria where porins that typically allow diffusion of antibiotics through the membrane are altered disallowing antibiotic entry; 3) Enhanced efflux of the antibiotic – efflux pumps are augmented or synthesized at faster rates to remove antibiotics from the intracellular space and cytoplasm before it can reach the target molecule; 4) Alteration of Drug Target – the mutation changes the structure of the target molecule so the antibiotic can no longer bind triggering its effect; 5) Pathway Alteration – This is the least common mutation due to its complexity, the bacterium produces an alternate redundant pathway mitigating the effectiveness of antibiotic blocking of the initial pathway.

One troubling element of resistance is how rapid it appears to be developing. For example in the 1940s when penicillin use was first becoming standard protocol for all isolates of S. Aureus <1% of isolates were resistant, but by 1951 approximately 75% of isolates were resistant which is why synthetic penicillin was created. For S. pneumoniae only 2% of isolates in 1981 were penicillin resistant versus 12% in 1995. It is thought that similar types of resistance development curves exist for other bacteria and other antibiotics. Of bacteria that have developed resistances the Infectious Diseases Society of America (IDSA) believes that Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, Enterobacter and E. coli are the most advanced and dangerous.11

The Gram-negative Acinetobacter, Pseudomonas, Enterobacter are becoming increasingly resistant to existing antibiotics at a much faster pace than their Gram-positive brethren. According to the CDC methicillin-resistance Staphylococcus aureus (MRSA) kills approximately 11,000 more than emphysema, AIDS, Parkinson’s or homicides.12 An additional 100,000 die from hospital acquired infections where a vast majority of those infections are resistant to at least one type of antibiotics.13,14

Antibiotics are misused primarily through excessive use of prophylactic strategies for travelers, failure to prescribe correct dosages through history of use and weight, failure to complete the prescribed dosage regardless of existing symptoms and inappropriate prescription of antibiotics for non-necessary conditions. In addition to misuse, the crisis among antibiotic resistant bacteria has largely come about due to a vast majority of pharmaceutical companies electing not to continue antibiotic research programs to develop new antibiotics. Not surprisingly without a continuous stream of new antibiotics, especially those with new core structures, increasing bacteria resistance has narrowed the available treatment options for existing bacteria infections. To highlight this deficiency in new antibiotics from 2008 to early 2013 only two systemic antibacterial agents were approved by the FDA versus the approval of sixteen from 1983-1987.14 Of at least 20 pharmaceutical companies that had large research and development programs in 1990 only AstraZeneca and GlaxoSmithKline remain today.15,16

One could argue that with the progression of time novel antibiotic discoveries become more difficult so it is understandable that fewer antibiotics would be developed now versus 20-30 years ago; while this rationality is correct a drop off of 87.5% in a 25-year span cannot be explained by such a rationality. In fact there are three principle reasons why a vast majority of pharmaceutical companies no longer focus on producing antibiotics.

First, there is little profitability in creating a new antibiotic largely because its application is single use over a short period of time versus the blockbuster (billion dollar+ per year) drugs that focus on chronic health or lifestyle conditions (statins, erectile dysfunction, high blood pressure etc.). In modern capitalism businesses prefer to sell a product that stabilizes a negative state/condition rather than cures it because the stabilizing agent can be sold in perpetuity. However, a counterpoint is that in the current market individuals pay $100,000 for an extra four months of life when facing stage-4 cancer, but balk at paying $125 for antibiotics that will help treat an infection. In this vein consumer priorities do appear misguided, based on fear rather than rationality ($100,000 for four more months and then death versus $125 for potentially decades of more life). This pricing difference is neither rational nor data-driven; there is no cost analysis that supports cancer drug pricing. Rather, drug pricing in the U.S. is based on public perception and fear. People are terrified of cancer, but not terrified by a multi-antibiotic resistant form of TB, which has much higher levels of virulence and a comparable fatality rate.

Second, drug research in general is expensive, especially now that most of the “low-hanging” fruit has been scooped off of the ground. The principle antibiotics that were first utilized in treatment (sulfonamides and penicillin) were derived from other species making their isolation and manufacture into a human applicable form straightforward. These compounds also provided effective core/lead compounds that could be slightly manipulated to produce new drugs, which had the potential to evade certain resistances bacteria developed for the initial principle compound. However, the manipulation of those core compounds has run fairly dry demanding more complexity, data mining and high throughput screening (i.e. computer power) to develop the next generation of antibiotics. Of course this complexity demands excess resources and financial capital increasing the costs of even attempting to create new antibiotics.

Third, some individuals believe that the regulatory system, especially in the United States, creates a sufficient obstacle for pharmaceutical companies. Critics cite that antibiotic approval by the FDA is confusing, unnecessarily complex and does not recognize the specialty of the antibiotic field of drug development versus other developmental fields. One of the more problematic elements of regulation is the narrow “approved for use” window assigned to antibiotics. For example when an anti-cholesterol drug is approved it is for broad use not high cholesterol values in the heart versus in the lower leg. When an antibiotic is approved it is approved to treat a specific single condition like pneumonia instead of the specific organism that the antibiotic or anti-bacterial targets.

Due to the volume based sales strategy with antibiotics and the narrow “approved for use” FDA rules where about one of every 72 new antibiotics are approved versus one of every 15 for other types of drugs, pharmaceutical companies would be encouraged to develop broad-spectrum antibiotics, which have higher resistance development profiles, the exact opposite of what antibiotic treatments should be striving for. Based on all of these elements: sales potential and regulation/research costs some estimate that in current market environment the production of a new antibiotic will actually cost a pharmaceutical company millions dollars over the life cycle of that given drug. Part of the problem with the additional regulatory steps demanded by the FDA could be what occurred after the approval of Telithromycin (brand name Ketek), which involved the use of fraudulent data to speed its approval resulting in numerous deaths and liver failures. Due to this incident the FDA has become more cautious when approving new antibiotics.

Passed in 2012 the GAIN act attempted to address some of the concerns with the deficiency in antibiotic research and development by extending marketing protections for select “qualified infectious disease products” by increasing levels of exclusivity (12 years to 17 years further delaying generics). Also there is an additional six months of exclusivity for drugs with a companion diagnostic test is approved. Basically this additional exclusivity tacks on to any Hatch-Waxman, orphan drug or pediatric exclusivity. This legislation also produced an expedited review awarding appropriate antibiotic candidates with priority review and fast track status.

In addition the FDA is responsible for revising guidelines for future antibiotic clinical trials to accurately reflect the latest scientific and clinical knowledge. So far in response to GAIN the FDA has created an anti-bacterial drug development task force to study future guidelines for drug development, released a list of high warning pathogens and reviewed with appropriate updates to numerous antibiotic drug development guidelines.

The problem with the GAIN act is it only focuses on one of the two important elements of the bacteria resistance issue, creating new antibiotics. There are no new regulations regarding the stewardship and application of existing and new antibiotics during the course of their administration. Without new rules new antibiotics will simply suffer the same fate as existing antibiotics and squander a percentage of their potential due to misuse.

Some may argue that FDA documents Final Guidance #209 and Draft Guidance #213 address this second element of usage and administration. However, voluntary recommendations are not effective rules and policy. #209 recommends that food/animal producers stop the use of medically important drugs (chiefly antibiotics) for growth promotion or feed efficiency purposes. It also suggests these producers seek veterinary oversight when application of any medical important drug is used for treatment or prevention. Not surprisingly it stands to reason that as only recommendations producers will only apply these actions if profitable. Seeing that a number of individuals/groups tend to ignore the antibiotic resistant problem and even violate existing usage law in the first place one cannot be confident that most producers will abide by these recommendations.

Draft Guidance #213 recommends that drug companies aid the application of Final Guidance #209 by removing any use suggestions on their drug labels pertaining to growth promotion. However, companies in the future will have the ability to list therapeutic uses, including disease prevention, on their labels. With antibiotic sales as a volume driven business it is difficult to see many companies voluntarily reducing the probability of sales of these products.

The FDA does acknowledge that making these guidelines voluntary is a significant handicap on their potential effectiveness, but claims that creating guidelines that have significant penalties associated with non-compliance will require time consuming formal hearings and legal processes that will more than likely see numerous evidentiary hearings and drag out for years partly because it would have to focus on one drug at a time . If this is the case then voluntary recommendations may be the best option to actually producing change.

In an attempt to combat the lack of new antibiotics the IDSA has proposed a specialized regulatory mechanism called the Special Population Limited Medical Use (SPLMU) exclusively for antibiotics that they wanted to be included in GAIN, but was not. The chief aspect of this new plan would change the approval process from two Phase III “lack of inferiority” trials to smaller Phase III trials using narrowly defined test subjects (those suffering from a condition born from a resistant bacteria). Despite the lack of safety information on these new antibiotic compounds it is thought that because these patients are already out of options due to the resistance the benefits derived from consuming the experimental compound will outweigh the risks. The thought is that through this direct treatment methodology safety and efficacy information can be derived for the experimental compound hastening the determination regarding whether or not it would be suitable for large-scale release.

This new testing regimen has been compared to the regimen utilized for development and approval of drugs for “orphan” conditions. However, orphan drugs will always remain a niche treatment area whereas these new antibiotics would eventually be used by millions of people with wide ranging characteristics that could produce a variety of side effects. Such a situation creates one of two strategies: 1) conduct secondary testing after initial approval to identify serious potential side effects and address any of these outcomes appropriately or 2) react and track new side effects when they occur in a broader public administration of the drug. Clearly the first strategy is more costly for it involves traditional Phase III testing, which in a sense is just postponed for a time after approval, but the second strategy creates legitimate questions of legal liability for any life altering side effects.

The two chief problems with this testing strategy are differentiating between drug related and bacterial related detrimental effects and off-label distribution. Phase III trials are tightly controlled, especially with regards to patient health, so effective rationalities can be made regarding the side effects associated with the tested drug and its usefulness in overall treatment. Direct treatment against very sick patients will create numerous problems determining whether future negative conditions are born from the experimental compound, the bacterium or a combination of both.

Off-label usage is a continuous problem as well. Despite the belief that specialized labeling, instructions to physicians and limited marketing should provide a sufficient barrier, the problem is that off-label usage is already widespread with existing drugs, so there is little to prevent the same behavior with these new experimental drugs. Very strict record keeping would be required including the IDSA suggested post-market surveillance plan. Off-label application should result in heavy sanctions against the offending party and if the sponsor company is responsible immediate cessation of the trial.

Some individuals have suggested that the future antibiotic research and development will depend on appropriate incentives to make the research and development economically sustainable. Some of the suggested incentives involve tax credits, special grants and improved intellectual property protections. While enhancing intellectual property protections is suitable and appropriate simply expecting the government to pay private pharmaceutical companies to develop antibiotics is not appropriate. What most individuals who clamor for direct financial incentives forget is that antibiotic development is direct income poor indirect income rich. In short a pharmaceutical company cannot sell their other drugs to a corpse, keeping the consumer alive is of utmost importance. Therefore, focusing on creating statins and other chronic no-cure (stasis) drugs over antibiotics is foolish because the consuming population will die making even the most profitable blockbuster drug not profitable. In addition most of the large pharmaceutical companies are not making “skin of their teeth” profit margins, thus to expect government, which is still running deficits, to incentivize antibiotic production is a short-term feasible long-term foolish strategy.

An interesting funding idea would involve all pharmaceutical companies that fail to develop new antibiotics to pay a royalty to those that do in effort to support antibiotic research and development. In addition this royalty would also allow successful companies to refrain from deployment of these new antibiotics until necessary. Furthermore this royalty could increase for novel mechanisms, which would also receive expedited approval from the FDA focusing on a new safety and efficacy track.

In addition to developing new antibiotics new usage policies need to be developed to ensure that these new antibiotics remain as fit as possible. There are numerous strategies that can and should be applied to limit the probability of resistance for new antibiotics. First, all nations must ban the use of antibiotics on non-human life despite the aforementioned reservations of the FDA. The shallow benefits provided by injecting mass quantities of antibiotics into healthy farm animals and the like are completely outweighed by the selective pressures these antibiotics provide for a more rapid development of resistance. The European Union has already done its part on this issue, it is time for the rest of the world to follow suit.

Second, the development of diagnostic and metabolic biometrics to create an “antibiotic floor” in order to prevent the foolish and unnecessary prescription of antibiotics to patients that should recover in a suitable fashion through standard non-antibiotic therapeutic strategies (hydration, sleep, exercise, etc.). This floor is important because it is more common than it should be for individuals suffering from viral infections to demand antibiotics that will do nothing but either be disposed of or increase resistance probability for current non-pathogenic bacteria residing in the patient. Third, sewage treatment methodologies should develop an additional step or agent that will increase the degradation rate for antibiotics in the waste stream or remove them entirely. Fourth, a standardized treatment methodology should be created to understand the timeline regarding the dosage and treatment pattern for how existing and new antibiotics attack a given infection. Establishing such a methodology will increase treatment rates and efficiencies and working in combination with the above second step should further limit over-prescription of antibiotics.

One longstanding idea regarding clinical trials in general is the lack of a centralized repository where clinical specimens from patients could be housed during and after clinical trials. These specimens would be used to reduce redundancy, increase efficiency in utilized dosage reducing costs and allow effective comparisons between drugs and/or control groups. This repository idea has also been proposed by the National Cancer Institute to retain samples from cancer patients undergoing various treatments.

Overall bacterial resistance is similar to global warming in the fact that it will have a significant detrimental influence on individuals and society as a whole, yet the global public at large does not view either as important problems that need to be solved in the near-future. Solving bacterial resistance will demand pharmaceutical companies stop being penny smart dollar stupid and realize that new antibiotics are essential to the profit maximization of other blockbuster chronic drugs. The FDA needs to realize that antibiotics require a specific pathway for approval and cannot simply be thrown into a generic approval pathway. Finally countries and companies have to work together to maximize information exchange to limit the spread of new resistant bacteria outbreaks and develop new treatments both through antibiotics and non-antibiotics to limit the probability of future resistance development.

Citations –

1. Walsh, C. Antibiotics: actions, origins, resistance. ASM Press; Washington, D.C: 2003.

2. Mukhtar, T, and Wright, G. “Streptogramins, oxazolidinones, and other inhibitors of bacterial protein synthesis.” Chem Rev. 2005. 105:529–42.

3. Tomasz, A. “The mechanism of the irreversible antimicrobial effects of penicillins: how the beta-lactam antibiotics kill and lyse bacteria.” Annu Rev Microbiol. 1979. 33:113–37.

4. Kohanski, M, Dwyer, D, and Collins, J. “How antibiotics kill bacteria: from targets to networks.” Nat. Rev. Microbiol. 2010. June: 8(6):423-435.

5. Nissen, P, et Al. “The structural basis of ribosome activity in peptide bond synthesis.” Science. 2000. 289:920–30.

6. Espeli, O, and Marians, K. “Untangling intracellular DNA topology.” Mol Microbiol 2004. 52:925–31.

7. Campbell, E, et Al. “Structural mechanism for rifampicin inhibition of bacterial RNA polymerase.” Cell. 2001. 104:901–12.

8. Food and Drug Administration. FDA's Strategy on Antimicrobial Resistance - Questions and Answers. http://www.fda.gov/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/ucm216939.htm

9. Coates, A, and Hu, Y. “Novel approaches to developing new antibiotics for bacterial infections.” British Journal of Pharmacology. 2007. 152:1147-1154.

10. Livermore, D. “Bacterial resistance: origins, epidemiology, and impact.” Clinical Infectious Diseases. 2003. 36(Suppl 1):S11-23.

11. Infectious Diseases Society of America’s (IDSA) Statement Promoting Anti-Infective Development and Antimicrobial Stewardship through the U.S. Food and Drug Administration Prescription Drug User Fee Act (PDUFA) Reauthorization Before the House Committee on Energy and Commerce Subcommittee on Health. March 8, 2012.

12. U.S. Department of Health and Human Services - Centers for Disease Control and Prevention. “Antibiotic Resistance Threats in the United States, 2013.” 2013.

13. Eisenstein, B, and Hermsen, E. “Resistant infections: a tragic irony in modern medicine.” APUA Clinical Newsletter. 2012. 30(1):11-12.

14. Centers for Disease Control and Prevention. “Estimating health care-associated infections and deaths in U.S. hospitals.” Public Health Rep. 2002. 122:160-166.

15. Choe, J. “Fewer drugs, more superbugs: strategies to reverse the problem.” APUA Clinical Newsletter. 2012. 30(1):16-19.

16. McArdle, M. “Resistance is futile.” The Atlantic. Oct. 2011. http://www.theatlantic.com/magazine/archive/2011/10/resistance-is-futile/8647/

Silver – Entering the real battle against resistant bacteria?

Due to the continuing progression of antibiotic resistant bacteria development in the medical community a number of individuals believe it is time to explore how to incorporate silver, a long standing antibacterial agent, into treatment strategies. On its face this appears to be an important new avenue for attacking the growing problem of resistant bacteria and the lack of development of new antibiotics to fight these bacteria. However, this excitement must be stemmed by appropriate knowledge of how silver helps fight bacterial infections and the potential pitfalls to its incorporation into actual antibiotics or other blood based treatment strategies versus its current medical uses as a sterilization and wound dressing tool.

The use of silver in medical and wound management dates back to the 18th century with the application of silver nitrate to treat ulcers.1,2 Anti-microbial activity for silver ions was first scientifically suspected in the 19th century, although anecdotal reasoning can be traced back much earlier.3 However, in the early 20th century the overall effectiveness of penicillin and later discovery of other antibiotics displaced silver use for over two decades. Silver use increased in the 1960s in response to burn treatments through the use of a silver sulfadizine (SSD) cream. In modern times silver is incorporated into wound dressings, antibiotic cream and first-aid plasters to reduce bacterial growth and limit infection.4,5

Both bulk silver and isolated silver nano-particles are effective at killing, not only standard Gram-negative and Gram-positive bacteria, but also antibiotic-resistant strains.6-8 This action also catalyzes antibacterial activity of current antibiotics including penicillin G, amoxicillin, erythromycin, clindamycin, and vancomycin.9 Various mechanisms have been suggested to explain the anti-microbial action of silver: 1) particle penetration of bacterial cell walls and membranes leading to DNA damage and apoptosis possibly due to free radical incursion; 2) alteration of bacterial cell wall and membrane, most likely targeting lipopolysaccharides, forming pits increasing membrane permeability for antibiotics or disrupting respiration for aerobes; 3) lowering probability of biofilm formation increasing the probability of antibiotic effectiveness.10-13

There is no clear evidence regarding whether the anti-microbial effects of silver are found in colloidal silver or ionic silver or both, but some evidence suggests that colloidal silver is much less effective as an anti-microbial agent than ionic silver, especially because only oxidized silver nanoparticles exert a significant antibacterial effect.14,15 If colloidal silver has anti-microbial action its magnitude is largely dictated by the dimensions of the particles where smaller particles have a greater effect due to higher surface area to volume ratios.16-18 The shape of the nano-particle also seems important with truncated triangles having superior activity.19,20

Ionic silver is thought to activate its anti-microbial effects through the formation of complexes with negatively charged sulfur, nitrogen or oxygen functional groups in bacterial enzymes destabilizing the bacteria and preventing normal functions like metabolism and reproduction.15,21 So silver nano-particles may simply be an inert element that eventually degrades creating the actual silver anti-microbial agent with degradation occurring faster at smaller sizes and certain shapes. Therefore, when studying anti-microbial activity and possible silver toxicity it stands to reason that one should focus on oxidized silver versus neutral silver.

While the anti-bacterial properties of silver were suggested in the 19th century, silver resistant bacteria and the basis for that bacterial resistance was first reported much later in 1975, first in Salmonella typhimurium.22,23 This resistance is chiefly born from plasmid development or transfer.24,25 The chief silver resistant plasmid has been labeled pMG101, is 180-182 kb with nine Open Reading frames (seven known and two unknown) in three transcriptional units and like most plasmids is conjugally transferable between bacteria.24,26 In addition to silver, pMG101 grants additional resistance to mercury, tellurite, ampicillin, chloramphenicol, tetracycline, streptomycin, and sulphonamide as well.23,24,27 Note the wide variety and importance of this resistance against not only silver, but other frequently used antibiotics. pMG101 presence can resist up to six times minimum silver lethality for E. coli and more than likely other bacteria species.24,27

Overall silver resistance is driven by encoding two silver efflux pumps (one an ATPase and the other chemiosmotic) and two periplasmic Ag+-binding proteins.26 Basically silver ion uptake is decreased and silver efflux is increased. There is also some belief that an ATP-dependent copper efflux protein can mediate removal of silver ions.1 However, there is no direct evidence that this silver resistance mechanism induces cross-resistance to various antibiotics, but the rate of cross-resistances to other antibiotics and genetic linkage of silver resistance and antibiotic resistance genes provides positive circumstantial evidence.

Various bacteria species that have been identified as having silver resistance are E. cloacae, Enterobacteriaceae, Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas stutzeri, P. aeruginosa and E. coli.27-35 Resistance is principally generated through sil genes (one of the genes contained in the pMG101 plasmid) with its functions having been assigned due to homologous genes that encode resistance to other genes.22 However, there is question regarding the total level of silver resistance provided by sil genes because some research has demonstrated only partial phenotypic silver resistance, thus providing only partial resistance to silver.22,28,29 The reason for this partial resistance could be driven by lack of complete transcription due to a lack of consistent silver forcing selection pressure. Finally silver resistance has developed from acinetobacter baumannii based on seemingly separate 54 kb plasmid (pUPI199).31

Despite the variety of bacteria that can acquire silver resistance, the high-level and multi-faceted mode of action from silver (cell wall destruction and reduced antibiotic efflux) lead most to believe that the development of silver resistant is unlikely. While silver resistant bacteria appear rare, one of the reasons for this rarity is thought to be that silver is not utilized at high levels for antibacterial purposes similar to current antibiotics and if this were to change so would the rates of silver resistance development.

One of the big problems with regards to determining an accurate accounting of silver resistance is the lack of standardized metrics for this resistance. There is disagreement on the MIC50 breakpoints for bacteria susceptibility.34 When most silver microbial susceptibility studies are performed the conducting researchers tend to assign their own breakpoints to delineate susceptible and resistant strains. For example results from studies that explored MIC values for Staphylococcus aureus ranged from 8 to 80 mg/L32,33 where studies for Pseudomonas aeruginosa produced a range between 8 and 70 mg/L.34,35

Another problem is a general lack of standardization for silver ion anti-microbial testing methods mostly driven by solubility issues that change the rate of available silver ions due to formation of various silver-halide anionic complexes.1 This influence is most noted in the sodium chloride content of the microbiological medium used for susceptibility testing.1 Also the lack of standardization in silver wound dressings creates numerous delivery systems to release silver ions, which include elements beyond simple silver concentration to influence the ability to kill microorganisms.36 These discrepancies create questions regarding how different tests can be compared for similarities or differences in method.

One of the principle concerns with incorporating silver into antibiotic treatments that directly enter the blood is the potential for serious negative side effects. There are three important factors that govern the ability of a metal to produce systemic toxic effect: 1) the rate of absorption due to the solubility of the metal; 2) the capacity to bind to biological targets in the body; 3) the residence time of the metal after absorption both at biological targets and in the blood stream before elimination through excretion. The form of silver that is introduced to the body influences these three elements. Currently silver is largely applied as either silver nitrate (inorganic silver salts) or as organic compounds like SSD.1

Normal (no medical or heavy industry work) silver concentration in the body is very low with <2.3 ug/L (or 3 nmol/L) in the blood, about 2 ug/day (or < 8 nmol/L) expelled in urine and 0.05 ug/g (wet tissue) in the liver and kidneys.37-39 Some evidence has demonstrated that some SSD treated burn patients have plasma silver concentrations as high as 50 ug/L after 6 hours of treatment and can reach a maximum of 310 ug/L.37 In one particular case a patient that died of renal failure after 8 days of treatment had silver concentrations of 970 ug/g, 14 ug/g and 0.2 ug/g in the cornea, liver and kidney respectively.37,38 Due to silver derived renal toxicity from topical application (from wound bandages) most physicians believe that it is improper to apply topical silver treatments over long periods of time even on burn patients.

However, despite the above information most believe that silver application is generally safe with accumulation occurring in superficial layers of the liver and kidneys with full clearance after 28 days.39,40 Such limited consistent binding and the rate of clearance implies superficial binding and low absorption from a single application. However, there is limited information regarding how organ absorption would change with direct silver application to the blood.

Overall there are still numerous safety and toxicity studies that must be performed before even considering applying silver directly to oral or intravenous antibiotic treatments. Also new policy limiting the application of ionic silver, in either direct or indirect form, to non-medical products for an anti-microbial agent must be created because mass application dramatically increases the probability for widespread resistance development. Such an outcome has already occurred for numerous existing antibiotics and based on the apparent resistance profile for sil genes and their corresponding plasmids the widespread development of silver resistant bacteria could create a pathogen that cannot be effectively managed through modern medicine.

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Citations –

1. Chopra, I. “The increasing use of silver-based products as antimicrobial agents –a useful development or a cause for concern?” Journal of Antimicrobial Chemotherapy. 2007. 59:587-590.

2. Klasen, H. “Historical review of the use of silver in the treatment of burns. I. Early uses.” Burns. 2000. 26:117-130.

3. Hugo, W, and Russell, A. “Types of antimicrobial agents. In: Russell, A, Hugo, W, Ayliffe, GAJ eds. Principles and Practice of Disinfection, Preservation and Sterilisation. Oxford, UK: Blackwell Scientific Publications: 1982. 8-106.

4. Fox, C Jr, “Silver sulfadiazine – a new topical therapy for Pseudomonas in burns. Therapy of Pseudomonas infection in burns.” Arch Surg. 1968. 96:184-188.

5. George, N, Faoagali, J, and Muler, M. “Silvazine (silver sulfadiazine and chlorhexidine) activity against 200 clinical isolates.” Burns. 1997. 23:493-495.

6. Burrell, R, et Al. “Efficacy of silver-coated dressings as bacterial barriers in a rodent burn
sepsis model.” Wounds. 1999. 11:64-71.

7. Yin, H, Langford, R, and Burrell, R. “Comparative evaluation of the antimicrobial activity of ACTICOAT anti-microbial barrier dressing.” J Burn Care Rehabil. 1999. 20:195-200.

8. Percival, S, Bowler, P, and Dolman, J. “Anti-microbial activity of silver-containing dressings on wound microorganisms using an in vitro biofilm model.” Int Wound J. 2007. 4:186-191.

9. Shahverdi, A, et Al. “Synthesis and effect of silver nanoparticles on the antibacterial activity of different antibiotics against Staphylococcus aureus and Escherichia coli.” Nanomedicine. 2007. 3:168-171.

10. Sondi, I, and Salopek-Sondi, B. “Silver nanoparticles as anti-microbial agent: A case study on E. coli as a model for Gram negative bacteria.” J Colloid Interface Sci. 2004. 275:177-182.

11. Hwang, E, et Al. “Analysis of the toxic mode of action of silver nanoparticles using stress-specific bioluminescent bacteria.” Small. 2008. 4:746-750.

12. Kim, J, et Al. “Anti-microbial effects of silver nanoparticles.” Nanomedicine. 2007. 3:95-101.

13. Morones-Ramirez, J, et Al. “Silver Enhances Antibiotic Activity Against Gram-Negative Bacteria.” Sci. Transl. Med. 2013. 5:190ra81.

14. Alvarez, J, et Al. “Negligible particle-specific antibacterial activity of silver nanoparticles.” Nano Lett. 2012. 12:4271−4275.

15. Lok, C, et Al. “Silver nanoparticles: Partial oxidation and antibacterial activities.” J Biol Inorg Chem. 2007. 12:527-534.

16. Baker, C, et Al. “Synthesis and antibacterial properties of silver nanoparticles.”
J Nanosci Nanotechnol. 2005. 5:244-249.

17. Panacek, A, et Al. “Silver colloid nanoparticles: Synthesis, characterization, and their antibacterial activity.” J Phys Chem B. 2006. 110:16248-16253.

18. Guzman, M, Dille, J, and Godet, S. “Synthesis of silver nanoparticles by chemical reduction method and their antimicrobrial activity.” Proceedings of World Academy of Science, Engineering and Technology. 2008. 33:367–74.

19. Pal, S, Tak, Y, and Song, J. “Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the Gram-negative bacterium Escherichia coli.” Appl Environ Microbiol. 2007. 73:1712-1720.

20. Wiley, B, et Al. “Shape-controlled synthesis of metal nanostructures: The case of silver.” Chem Eur J. 2005. 11:454-463.

21. Damm, C, Munstedt, H, and Rosch, A. “Long-term antimicrobial polyamide silvernanocomposites.” J Mater Sci. 2007. 42:6067–73.

22. Woods, E, Cochrane, C, and Percival, S. “Prevalence of silver resistance genes in bacteria isolated from human and horse wounds.” Veterinary Microbiology. 2009. 138(3-4):325-344.

23. McHugh, G, et Al. “Salmonella typhimurium resistant to silver nitrate, chloramphenicol, and ampicillin.” Lancet. 1975. 1(7901):235-40.

24. Gupta, A, Maynes, M, and Silver S. “Effects of halides on plasmid-mediated silver resistance in Escherichia coli.” App. Environ. Microbiol. 1998. 64(12):5042-5.

25. Davis, I, Richards, H, and Mullany, P. “Isolation of silver- and antibiotic-resistant Enterobacter cloacae from teeth.” Oral Micro and Immunol. 2005. 20(3):191-4.

26. Silver, S. “Bacterial silver resistance: molecular biology and uses and misuses of silver
363 compounds.” FEMS microbiol rev 2003. 27(2-3):341-53.

27. Gupta, A, et Al. “Molecular basis for resistance to silver cations in Salmonella.” Nature Med. 1999. 5(2):183-188.

28. Annear, D, Mee, B, and Bailey, M. “Instability and linkage of silver resistance, lactose fermentation, and colony structure in Enterobacter cloacae from burn wounds.” J. Clin Pathol. 1976. 29:441-443.

29. Hendry, A, and Stweart, I. “Silver-resistant Enterobacteriaceas from hospital patents.” Canadian J. Microbiol. 1979. 25:915-921.

30. Bridges, K, et Al. “Gentamicin and silver-resistant Pseudomonas in a burns unit. Br. Med. J. 1979. 1:446-449.

31. Deshpande, L, and Chopade, B. “Plasmid mediated silver resistance in Acinetobacter baurmannii.” Biometals. 1994. 7:49-56.

32. Ug, A, and Ceylan, O. “Occurrence of resistance to antibiotics, metals and plasmids in clinical strains of Staphylococcus spp.” Arch. Med Res. 2003. 34:130-136.

33. Hamilton-Miller, J, Shah, S, Smith, C. “Silver sulphadiazine: a comprehensive in vitro reassessment.” Chemotherapy. 1993. 39:405-409.

34. Vassishta, R, Chhibber, S, Sexena, M. “Heavy metal resistance in clinical isolates of Pseudomonas aeruginosa.” Folia Microbiol (Praha). 1989. 34:448-452.

35. De Vicente, A, et Al. “Resistance to antibiotics and heavy metals of Pseudomonas aeruginosa islated from natural waters.” J. Appl Bacteriol. 1990. 68:625-632.

36. Thomas, S, and McCubbin, P. “A comparison of the anti-microbial effects of four silver-containing dressings on three organisms.” J. Wound Care. 2003. 12:101-107.

37. Wan, A, et Al. “Determination of silver in blood, urine, and tissues of volunteers and burn patients.” Clin Chem. 1991. 37(10 Pt 1):1683–1687.

38. Boosalis, M, et Al. “Serum and urinary silver levels in thermal injury patients.” Surgery. 1992. 101(1):40–43.

39. Coombs, C, et Al. “Do burn patients have a silver lining?” Burns. 1992. 18(3):179–184.

40. Harrison, H. “Pharmacology of sulfadiazine silver. Its attachment to burned humans and rat skin and studies of gastrointestinal absorption and extension.” Arch Surg. 1979. 114(3):281-285.