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Alternatives to Antibiotics
April 17, 2009
Suzie Fisher, Ethan Fry, Quynh Pham
and Micah Tobin
Antibiotics today, like penicillin, are composed of a variety of proteins that generally interfere with or kill bacteria. While the use of these drugs has improved anti-bacterial therapy and has been a successful treatment method, it has also led to the development of bacterial resistance and the creation of superbugs like MRSA (methicillin resistant staphylococcus aureus), VRSA (Vancomycin resistant staphylococcus aureus), vancomyin resistant E. faceium, and others with multiple drug resistance. New technologies are emerging that can help combat these types of infections like therapeutic phage therapy (the use of anti-bacterial viruses, which aren’t really new, but certainly re-emerging), direct use of bacterial cell wall hydrolases, and the use of a variety of other antimicrobial peptides. These methods can revolutionize patient treatment since they bypass antibiotic resistance. They could also redefine the function of a clinical microbiologist, with the introduction of new methods of bacterial identification. In addition, these treatments would require laboratory monitoring to determine effectiveness of phage/hydrolase treatment.
Several mechanisms by which VRSA exhibits antibiotic resistance.
The evolution of MRSA is an excellent example of how quickly problems can arise with drug resistance. By 2003 nearly 50% of the S. aureus recovered in hospitals were resistant to both penicillin and methicillin1. MRSA then started exhibiting resistance to glyopeptides and had low level resistance to vancomycin. Shortly thereafter there emerged highly resistance strains of MRSA. VRSA also shows resistance to a variety of other drugs: clindamycin, aminoglycosides, trimethoprim–sulfamethoxazole, rifampin, and fluoroquinolones Other organisms, as well, are a cause of great concern such as penicillin and vancomyacin 'tolerant' enterococci, vancomyacin and ampicillin resistant Enterococus faceium, multidrug resistant P.aeruginosa and Acinetobacter, as well as beta-lactamase (enzyme that hydrolyzes the beta-lactam ring of penicillins) producing Klebsiella, E. coli, and Enterobacter. Alarmingly, the gene that codes for the production of beta-lactamases are often part mobile genetic elements and able to quickly spread horizontally to other bacteria.
Unfortunately the development and distribution of new antibiotics has not kept pace with the evolution of bacterial resistance. In fact only two new classes of antibiotics of novel mechanisms of action (linezolid and daptomycin) have been introduced into the market during the last three decades. In addition, these two new classes of drugs have important toxic side effects1.
A bacteriophage, or 'phage' for short, is a greek term that translates as a bacteria-eater. They are viruses that infect and kill bacterial cells, using them to replicate more viruses. dsDNA tailed phages, or "Caudovirales", account for 95% of all the phages reported in the scientific literature. They, like human viruses, take over the genetic machinery of a cell using injected RNA and DNA, and will enter either a lytic or lysogenic phase (depending on the virus).
Electron micrograph of bacteriophages attacking a bacterial cell.
Phages that induce the lytic phase are those useful for phage therapy11. Bacterial cell walls are broken and destroyed, whereupon replicated virions are released. Lysogenic phages (sometimes called temperate phages/prophages) integrate their injected DNA into a host cell chromosome or plasmid. This DNA will be present in all of the cells offspring. They will eventually lyse the cell when the host cell’s conditions deteriorate. Sometimes however, these prophages benefit the host because the DNA that they insert into the bacterial genome could contain a gene coding for antibiotic resistance (for example, if that virus came from a recently lysed bacterial cell that had a gene that had antimicrobial effects). Evidence suggests that the spread of antibiotic resistance has been accelerated by prophage infection15. There is current research investigating the introduction of clustered, regularly interspaced, short palindromic repeat (CRISPR) loci inpathogenic bacteria that can limit the prophage functionality, thereby further enabling the use of bacteriophages as therapy agents14.
Phage were used as early as the 1020's in both Russian and European medical groups as antimicrobial therapy, and were relatively successful17. They were able to successfully treat a variety of bacterial conditions. To pursue this goal the Eliava and Hirszfield Institutes was created and continues to research phage therapy. However, the emerging success of penicillin and other antibiotics eclipsed the use of phages. Unfortunately, bacteria have evolved to exhibit resistance to these antibiotics and now we have run full circle, approaching a pre-penicillin era.
Bacteriophages are useful as antimicrobial agents for a variety of reasons including: they are unaffected by antibiotic resistance, the ability to penetrate biofilm, non-induction of shiga toxin production, usefulness as identification agents, and research indicating phages to be highly effective with minimal side effects.
With the rise of drug resistance bacteria and the lack of useful new antibiotic drugs the fact that bacteriophages are bypass antibiotic resistance, (ie. current resistances to antibiotics are due to mechanisms that will have no effect on a bacteriophage's ability to infect bacterial cells) is quite alluring. Phage resistance is also not developed on a broad scale, and targeted bacteria would remain susceptible to other similar target phages. This differs from antibiotic therapy in that antibiotics have a broad range and select for many drug resistant species, increasing the overall resistance17.
Current research also indicates that phages have the capability to penetrate bacterial biofilm7,8, and when used in conjunction with antibiotics can help to deliver the antibiotics more effectively to the targeted bacteria3. This is important because BRI's or biofilm related infections have been a major barrier for treatment since conventional antibiotics are unable to reach the bacterial organisms. In addition, phages have been engineered to induce the bacterial cell to produce depolymerases that degrade the biofilm.
One potentially groundbreaking find is that in contrast to antibiotics, phage treatment of pathogenic E. coli strains did not induce Shiga toxin production19. This is probably related to the phages ability to lyse (form plaque on) a high percentage of pathogenic E. coli strains of various serotypes and origins, while displaying low lytic (plaque-forming) capacity on non-pathogenic ECOR strains. This could have major implications on treatment of all toxin producing bacteria.
Another use for phages is bacterial identification, which is a very important first step toward treatment. Currently a variety of cultures, chemical tests, and antimicrobial susceptibility testing is used. Phage susceptibility testing can now be included in that list as well and is very useful because phages are highly specific, which can be a detriment as well (susceptibility testing could only be done in the later stages of identification due to the large number of possible bacteriophages)5,13,18.
The first bacteriophage clinical trials took place as early as 1919 and, even though research subsequently focused more on antibiotics, many more clinical studies have been done, including animal and human trials. One quite recent animal study utilized treatment of klebsiella by bacteriophages in mice and guinea pigs. Treatment was administered in a variety of doses, which reached up as high as 3,500 times the human estimated dosage, with no adverse side effects and successful treatment of the infection. In addition, extensive human trails17 have been performed (mostly in Poland and Russia) using numerous organisms, they also indicate very little, if any, negative side effects. One of these trials dates back to 1963 and involves a widescale outbreak of bacterial dysentary that was successfully treated using shigella phages. Another lengthy set of research papers done in Poland involved over 550 patients that had septicemic infections of Staphylococci, Pseudomonas, Escherichia, Klebsiella, and Salmonella. In it, they reported a 75-100% overall success rate and a 94% success rate with patients that didn't respond to antibiotics. Further clinical trials have been done on high risk patients like those with cancer or with a compromised immune system, all with minimal side effects4,6.
There are several problems associated implementing therapeutic use of bacteriophages. 1) Is that not all bacteria have a corresponding phage that could be used for treatment. 2) They are highly specific. This is both good and bad in that it means low disruption of normal flora and low chance of secondary infection, but also means if identification is not exactly right, the phages will not be effective. 3) Packaging and administration of treatment is a focus of current research, but at this point demonstrates somewhat poor viability and stability. 4) The development of phage-neutralizing antibodies by the host. While the occurrence of this phenomenon is well documented, it is unclear how significant of an effect it has on phage therapy1,17.
Despite the drawbacks that clinical use of bacteriophages presents, they clearly have a therapeutic edge over current antibiotics and are an attractive option as an alternative for conventional antibiotics.
Bacterial cell wall hydrolases are enzymes that degrade peptidoglycan, which is the major component of the bacterial cell wall, and cause bacteriolysis. They target the integrity of the cell wall and they are designed to attack major bonds in the peptidoglycan. 21
Endolysins are double-stranded DNA bacteriophage-encoded peptidoglycan hydrolases. They were referred as Virolysins.24 They are produced in phage-infected bacterial cells toward the end of the lytic cycle. Lysins are not lytic enzymes. Lysins are capable of digesting the cell wall when used as recombinant proteins. Endolysins are divided into five main classes: N-acetylmuramidases (lysozymes), endo-B-N-acetylglucosaminidase, lytic transglycosylases. These three cleave the sugar moiety of peptidoglycan. Endopeptidases cleave the peptide moiety. N-acetylmuramoyl-L-alanine amidases cut the amide bond between both moieties.20 Lysozyme-like enzymes (muramidases) hydrolyze N-acetylmuramyl-1,4-ß -N-acetylglucosamine bonds. Lytic transglycosylases act on the same bond as muramidases, but further catalyze the intramolecular transfer of the O-muramyl residue to its own C-6 hydroxyl group. N-acetylmuramyl-L-alanine amidases hydrolyze the bond between N-acetylmuramic acid and L-alanine.26
Lytic enzymatic activities; attacking specific sites in the peptidoglycan network, leading to peptidoglycan hydrolysis and bacteriolysis. It will first bind to specific sites on the bacterial cell wall, then cleave specific peptidoglycan bonds.
Nonlytic mechanism: provoke perturbation or to activate the autolytic system of bacteria, it is based on the cationic and amphiphilic properties of bacterial cell wall hydrolases.24
Lysins lack secretory signals, they are not translocated through the cytoplasmic membrane to attack their substrate in the peptidoglycan. They depend on holins (small hydrophobic proteins) to get to peptidoglycan from inside the cell. Holins are the second phage gene product in the lytic system.21 These holins enable endolysin molecules to cross the inner membrane.24 Holins are produced during phage infection cycle within a host organism to perforate the bacterial membrane. Endolysins then cleave covalent bonds in the peptidoglycan, leading to the lysis of the bacterial cell.23
Below is the cycle of phage lysin:
The images below are electron micrographs showing phage enzyme wiping out a colony of Bacillus cereus. They demonstrate (a) a healthy colony of bacteria; (b) the bacteria 1 minute after enzyme treatment; (c) the bacteria after 15 minutes of enzyme treatment; (d) lysin punching holes in bacterial outer cell wall, causing it to explode; (e) a single bacterium beginng to rupture (focus on the arrow, it shows the inner membrane spilling through an enzyme-induced hole) 25
BCWH: Pros and Cons
Lysins have a novel mode of action: selective and rapid killing of pathogenic bacteria with no effect on the normal microflora. As a result, there are no side effects or secondary infections such as those that occur in treatment with antibiotics. 22
They have a narrow antibacterial spectrum: unique linkages to be cleaved in the cell wall, specific enzyme activation by components present exclusively in or on the cell wall, specificity in substrate recognition and cell wall binding.
BCWH demonstrate activity against baceria regardless of antbiotic senstivity.
Phage lysins have a low probability of developing resistance.20 Lysins are known as "intelligent" drugs due to the fact that they multiply at the site of the infection until there are no more bacteria, then they are excreted. 22
There has been only one report about the activity of an endolysin compared with an antibiotic. Currently, there is no general conclusions that can be fully drawn that show that lysins are more effective than any traditional antibiotics.20
Lysins only work with gram-positive bacteria, since they are able to make direct contact with the cell wall carbohydrates and peptidoglycan when added externally, whereas the outer membrane of gram-negative bacteria prevents this interaction.
A little over twenty years ago it was discovered that frog skin, lymph of insects, and neutrophils from human blood all contain cationic peptides that have distinct antimicrobial abilities. As antibiotic resistance became a growing concern among the health professionals, a novel theory was thrown around, and that theory involved research into our “evolutionary ancient weapons”.31 These ancient weapons have been around as long as the evolving multicellular organism and are still utilized today, by our immune system. Antimicrobial peptides (AMPs) are natural defenses found in all types of living organisms. These peptides are encoded by genes, and the study of them in the past two decades have shown promising results as alternatives to our current antibiotics. AMPs are cationic chains of peptides that utilize their hydrophobicity to react and disrupt cell membranes allowing them to act as innate barriers to infection.27 The search for antibiotic alternatives has been ongoing for decades but it wasn’t until the late nineties that AMPs began to be fully understood and their therapeutic activity was realized. Two classes were discovered in the late nineties; the first was a ribosomally-synthesized peptide and the second was a non-ribosomal synthesized peptide found in bacteria, lower eukaryotes, and plants.29 Since then, research has been ongoing and over 1300 AMPs have been identified and can be found in the Antimicrobial Peptide Database (ADP2). This includes 65 anti-cancer, 76 anti-viral, 327 antifungal and over 950 antibacterial peptides classified.30
Many different AMPs have been discovered throughout the years and as they were discovered they were classified mainly on the basis of their biochemical charge (some are anionic) or structural features (linear/circular/amino acid composition). Aside from the ADP classifications these classes of AMPs have been formed:29
Cationic Peptides = Largest group of AMPs. Over 50% of this type have been isolated from insects. There are 3 groups of cationic peptides:
1- Linear peptides forming helical structures
2- Cysteine-rich open-ended peptides with disulfide bridge(s)
3- Amino acid rich molecules
Subfamilies of cationic peptides:
Cecropins--Family of 3-4 kDa linear peptides. First described in the 1980s they provided the first research into their biomedical applications.
Defensins- -Family of 4 kDa open-ended cysteine-rich peptides with several motifs. They are characterized by their unique structure involving an a-helix linked to a b-sheet by two disulfide bridges.
Thionins - -Generally basic plant peptides with a molecular weight of 5 kDa.
Amino acid-enriched - -Large class that differ from one another due to the different amino acid within the structure. This class has broad spectrum activity against hundreds of gram negative and positive bacteria, fungi and even the enveloped HIV virus.
Histone-derived - -Family that is structurally similar to cecropin and especially active against bacteria and fungi. Mode of action involves penetrating the membrane and binding to the nucleic acids leading to cell death.
Beta-hairpin - -Class of 2-8kDa structures that contain a beta-hairpin cross-linked by disulfide bridges. Varying numbers of bridges within the class. Antibacterial and antifungal capabilities.
Anionic Peptides-- Smaller class than cationic peptides and mostly isolated from mammalian species.
Neuropeptide derived - -First class of anionic compound to be found. Active against gram-positive bacteria at micromolar concentrations.
Aspartic acid-rich - -Isolated mostly from cattle pulmonary surfactants. Also seen in human sweat and is active against gram-positive bacteria.
Aromatic dipeptides - -Not much is known at this point. Derived from different fly species.
Oxygen-binding proteins - -Bactericidal compounds that aren ’t as potent as the cationic peptides, but could complement the activity of the other compounds. Physiological relevance remains to be established.
Prokaryotic OriginThe AMPs produced by bacteria have been grouped into many different classes based on criteria such as their producer organism, molecular size, and chemical structure. The most relevant are produced by gram-positive bacteria and are a part of the bacteriocins class. Bacteriocins range in size from 1.9 to 5.8 kDa and can be cationic, neutral, and anionic in their chemical makeup.
- Cell lysis, though not probable due to cationic peptide concentration
- Channel formation through reorientation of the peptides within the membrane
- Carpet model where the peptides break down the membrane integrity
- Killing of bacterial cell by peptides attacking internal targets31
Escherichia coli treated with an antimicrobial cationic peptide at low concentration (top)and high concentration (bottom). Note the formation of blebs (small spheres of membrane)
that are coming off of the bacterium, as the membrane is damaged.