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Pseudomonas aeruginosa {BioBrick BBa_K1683005 & BBa_K1683004}

Quick Facts:
Gram (-)
Aerobic
Extracellular infection

Types of infections that it causes:
  • Infecting burned tissue and causing lung infections in people with cystic fibrosis.
    • In burned tissue, the wound seeps plasma, rich in nutrients. P. aeruginosa then takes the opportunity of this nutrient rich “media”. We must fight these infections quickly, because this strain is notorious for its antibiotic resistance. Once it spreads to the bloodstream, it becomes even more challenging to fight, and this is what can cause death in patients.
    In cystic fibrosis, infection is slower. Infection occurs in 80% of cystic fibrosis patients within the first few years of their life. [bacterial pathogenesis]. Once it exists in the lungs, antibiotics can help slow the infection, but eventually bacteria develop physiological traits that increase resistance, such as the alginate capsule.
  • Skin infections
  • Bloodstream infections
  • Urinary tract infections
  • Wound infections
  • Nosocomial (hospital acquired)

Infections are rare even though it has a widespread distribution. It inhabits wet surfaces, especially hospital surfaces, which contributes to its nosocomial traits. P. aeruginosa is originally a soil microorganism.

Virulence Factors:
P. aeruginosa has many virulence factors. They are categorized into physical characteristics, such as nonpilus adhesions and flagella. There are also enzymes that function as toxins. These virulence factors work to damage the surface and membranes of eukaryotic membranes. Pyocyanin is a cytotoxin that interferes with the a normal redox reaction in host cells, creating reactive oxygen. This damages our cells and causes tissue necrosis. In addition, it produces rhamnolipids, which form a barrier around the cell known as “shielding”. This barrier protects the cell against leukocytes, further barring our immune system from fighting the infection. P. aeruginosa are also known to quorum sense and form biofilms, making the infection yet harder to treat. Yersinia pestis {BioBrick BBa_K1683005 & BBa_K1683004}

Quick Facts: Gram Negative
Nonmotile
Anaerobic
Obligate Parasite
Course of Infection
Yersinia pestis is the cause of what is colloquially known as the Plague. Primarily found in rodents, which contract the bacteria from their frequent contact with fleas, humans play no role in the long term survival of Y. pestis. (Perry 51) Transmission of the bacteria occur when fleas feed on the blood of rodents, some of which is regurgitated into the mammal. The spread then continues through predators and other mammals that either come into contact with infected rodents or directly ingest the infected rodent. Today, domesticated cats are the primary cause of most Y. pestis infections, prompting concerns over veterinarian health and creating a resurgence of attention for the bacterium.

Infection Types
There are three primary forms that human plague cases will occur in, these being bubonic, septicemic, and pneumonic. “Over the past 24 years, the majority of patients in the United States have had either bubonic or septicemic plague.” (Perry 57) Pneumonic plague is far more rare and deadly. It is spread by respiratory droplets and its incubation period can be as little as one to three days. Symptoms of the bubonic form include painful and swollen lymph nodes and fever. The symptoms of the other two forms are hard to detect. Septicemic plague symptoms resemble that of other gram-negative septicemias (Perry 58) and pneumonic plague can be misdiagnosed as other pulmonary infections since chills, headaches, and gastrointestinal disturbances are common symptoms.

Treatment
All three forms are treatable with antibiotics such as Streptomycin, Gentamicin, Oxytetracycline, Tetracycline, Chloramphenicol, and Doxycyline. Streptomycin is the antibiotic of choice for most treatments but due to its high toxicity its use is usually stopped after five to ten days and switched with less toxic antibiotics. Upon diagnosis, immediate isolation is required for at least two days to allow antibiotic treatment to take effect.

Virulence Factors
  • Some of the most common virulence factors of Y. pestis include serum resistance, the Fraction 1 capsule and murine toxin.
  • Serum resistance is the ability of Y. pestis to resist complement-mediated lysis since it must be able to survive (and/or grow) in blood; the primary mean of infection. An integrated pMT1 gene allows for complete resistance to complement lysis. Y. pestis is resistant even in the presence of antibodies and is chromosomally encoded. The rough or short lipopolysaccharide of Y. pestis appears to mediate this resistance.
  • Fraction 1 is the name given to a potent antigen of the human blood stream. Unfortunately, Y. pestis possesses the ability to form a large gel like capsule around itself to prevent the antigen from binding to it. This form of resistance also coincides with Y. pestis’ ability to resist phagocytosis.
  • Murine toxin is generally toxic in mice and rats but is relatively useless against larger mammals such as dogs, monkeys, and chimpanzees. The protein responsible is coded for by the kDa gene on the 110-kb plasmid of Y. pestis.

Robert D. Perry, J. D. (1997). Yersinia pestis-Etiologic Agent of Plague. Clinical Microbiology Reviews, 35-59. Retrieved 2015, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC172914/pdf/100035.pdf





Antibiotic definition:

Antibiotics literally mean “agents against life.” Thus they are compounds which inhibit the growth of (bacteriostatic) or kill bacteria (bactericidal) under appropriate concentrations. Some are produced naturally by various organisms while others are produced synthetically in the laboratory. Ideally an antibiotic causes minor, if any, side effects to humans and animals, has a broad spectrum of activity which will be effective against many kinds of bacteria, and bioavailability to effectively reach and act in the area of bacterial infection.(1)

Cell membrane differences:

Bacteria are commonly classified according to the structure of their cell membranes: gram positive, gram negative, acid-fast, and mycoplasma. Most bacteria fall into either the gram positive or gram negative category.(2)

For a gram positive bacterium, the interior of the cell is surrounded by a typical phospholipid bilayer. Outside this cell membrane is a thick layer of peptidoglycan, or murein, which is a polymer of sugars and amino acids. This murein covering is responsible for giving structure and shape to the bacterium, gives resilience to osmotic pressures, and provides an effective defense mechanism against antibiotics or other harmful chemicals. Techoic acids are other polymers which adhere to the outside surface of the murein layer of gram positive bacteria. Some bacteria also have an outer capsule encompassing the whole cell.(2)

The structure of the gram negative cell membrane is more complex. Rather than simply constructing the thick murein layer typical of gram positive bacteria, gram negative bacteria utilize a thin layer of murein surrounded by another outer membrane. The interior of the cell is surrounded by the typical phospholipid, inner membrane. A relatively thin layer of murein lies outside the inner membrane and a unique outer membrane envelopes the murein. The resulting space between the two membranes is called the periplasm, or periplasmic space. The fluid of the periplasm contains a variety of proteins. Many of these proteins are binding proteins or enzymes for metabolism. Some gram negative bacteria also have β–lactamases to inhibit antibiotics in the periplasmic fluid. The outer membrane is bilayered, but is distinctive for the lipopolysaccharide (LPS) molecules found in the outermost layer. The cell membranes of gram negative bacteria must also have porins, or channels, to allow the entry of small (700 daltons or less) essential hydrophilic compounds while larger ones require specialized transport mechanisms. Thus gram negative cells have a defense system of degradative enzymes in the periplasm and porins and specialized transport mechanisms exclusively for essential hydrophilic molecules.(2)


http://www.nature.com/nrd/journal/v2/n8/images/nrd1153-f1.jpg

Antibiotic mechanisms:

The majority of utilized antibiotics target one of five vital processes in bacterial growth: cell wall synthesis, protein synthesis, DNA synthesis, RNA synthesis, or tetrahydrofolate synthesis.(1)

Cell wall synthesis inhibitors include β-lactam antibiotics, glycopeptides, and others. The β-lactam antibiotics are so named for the four-member ring structures that they have in common. Examples of β-lactam antibiotics include the penicillins, cephalosporins, carbapenems, and monobactams. These β-lactam antibiotics function to inhibit cell wall synthesis by preventing the transpeptidation reaction responsible for linking the peptidoglycan, or murein, side chains together. β-lactams may also covalently bond with the transpeptidase enzyme which similarly inhibits cell wall synthesis and triggers autolysins which normally degrade peptidoglycan during bacterial growth and division. The glycopeptides include vancomycin, daptomycin, and teichoplanin which are especially effective against gram positive bacteria. This is because they inhibit the final steps of peptidoglycan synthesis by binding to the D-Ala-D-Ala muropeptides thereby preventing the essential cross-linking of the peptidoglycan. Other antibiotics like fosfomycin and bacitracin inhibit cell wall synthesis at more initial stages, specifically the conversion of N-acetylglucosamine to N-acetylmuramic acid. Without intact peptidoglycan, the bacterium is more likely to lyse from osmotic pressure with its environment.(1)

Protein synthesis inhibiting antibiotics include aminoglycosides, tetracyclines, macrolides, and lincosamides. These antibiotics exclusively interrupt bacterial translation by taking advantage of the structural differences between prokaryotic and eukaryotic ribosomes. Aminoglycosides such as kanamycin and gentamicin are trisaccharides with amino groups. They bind to sites of the 30S subunit of the prokaryotic ribosome thus preventing its union with the 50S subunit to create a fully functional ribosome. Tetracyclines similarly bind to the 30S subunit of the ribosome, but they distort the A site such that aminoacylated tRNA cannot interact properly with the mRNA transcript. The macrolides, such as erythromycin, impede translation by binding to the 50S ribosomal subunit such that the E site is blocked. The lincosamides which include lincomycin and clindamycin function similarly to the macrolides but have different structures. Inhibition of protein synthesis tends to be bactericidal.(1)

DNA synthesis inhibitors include the quinolones such as nalidixic acid and the fluoroquinolones such as norfloxacin and ciprofloxacin. These antibiotics interfere with DNA gyrase which is essential for relaxing the positive supercoils that results during DNA replication, recombination, and transcription. Without functioning DNA gyrase, DNA becomes irreparably broken and dysfunctional. Another antibiotic, metronidazole, requires activation by flavodoxin but can then causes damage directly by nicking DNA. Intact DNA is essential for continued growth and life of an organism.(1)

Rifampin exemplifies antibiotics that inhibit RNA synthesis. It inhibits transcription by binding to the β subunit of RNA polymerase. It is analogous to macrolides in that rifampin sterically blocks the exit of the RNA polymerase as macrolides block the exit site of the ribosome during translation. RNA is the essential intermediate in the central dogma of molecular biology; without it proteins cannot be synthesized and the organism will die.(1)

The antibiotics trimethoprim and sulfonamides inhibit the tetrahydrofolate biosynthesis pathway in bacteria which is necessary for the synthesis of amino acids including fMet. These antibiotics inhibit the enzymes required to produce tetrahydrofolic acid which is necessary for the reactions that result in the formation of nucleic acids and fMet. Mammals do not synthesize tetrahydrofolic acid which decreases the risk of unwanted side effects in their application. These antibiotics are generally only bacteriostatic because synthesis of new amino acids is not absolutely necessary.(1)

This is not an exhaustive list, but merely an overview of the main groups of antibiotics. There is a need for new antibiotics, both discovered in nature and synthesized in the lab, to effectively combat infectious bacteria which continue to evolve mechanisms of defense.


https://upload.wikimedia.org/wikipedia/commons/0/08/Antibiotics_Mechanisms_of_action.png

Modes of resistance:

Bacteria have evolved methods to be resistant to various antibiotics. Resistance differs from tolerance in that it causes the bacterium to stop growing while the concentrations of antibiotic are high, it does not require mutations or additional genes, and it is reversible. A resistant bacterium will grow even in the presence of antibiotic, gained resistance from mutation or from a resistance gene, and is not reversible.(1)

Mechanisms of antibiotic resistance tend to fall into one of four general areas: preventing the antibiotic from reaching its target (often by efflux), chemically modifying the antibiotic so it becomes harmless, modifying the antibiotic’s target so it is unaffected by the antibiotic, or preventing the antibiotic from becoming activated.(1)

There are several ways that bacteria prevent an antibiotic from reaching its target and taking effect. Merely the structure of the cell membrane, particularly in gram negative cells forms the first line of defense. Antibiotics must pass through the outer membrane and the inner membrane to reach the cytoplasm. There are outer membrane porins which allow for transport of some molecules, but they selectively permit certain molecules based on size. Large, bulky molecules are unable to enter the cell through these porins. However mutations in the genes for the porin proteins can act either to broaden or narrow the opening of these porins thus changing the selection of which molecules can enter the cell. Antibiotics may also be prevented from crossing the cytoplasmic membrane. A more common resistance mechanism is the active efflux pump. Efflux pumps are ubiquitous in bacteria and work to prevent the accumulation of an antibiotic in the cell so that it never reaches an inhibitory concentration. These pumps may be specific or compatible with many substrates and are found in both gram positive and gram negative bacteria. Efflux pumps have been reported for every group of antibiotic, causing a significant challenge in combating bacterial infection.(1)

Bacteria may also enzymatically inactivate an antibiotic, rendering it harmless. Gram negative bacteria commonly have β-lactamases in the periplasm which hydrolyze the antibiotic to cleave C-N bond in the ring structure. Sensitivity to β-lactams antibiotics has been partially regained by combining the β-lactam antibiotic with a β-lactamase inhibitor such as clavulanic acid or sulbactam. Unfortunately some bacteria have already become resistant to this method as well. An additional technique bacteria use to combat antibiotics is to modify them with the addition of a chemical group. Phosphorylation, adenylation, and acetylation of a hydroxyl or amino group may be sufficient to interfere with the activity of the antibiotic. This is the primary form of aminoglycoside resistance which would otherwise act to prevent protein synthesis. Similarly acetylation of chloramphenicol and streptogramin render these antibiotics ineffective. Tetracyclines can also be inactivated by an oxidation reaction.(1)

Another strategy to resist antibiotics is the altering or defending the target of the antibiotic usually by mutations that change the antibiotic’s target or by chemically altering the target with the addition of chemical groups. β-lactams are also sensitive to this form of resistance when mutations cause the binding specificity of the penicillin binding proteins to change. Even resistance to vancomycin which targets the D-Ala-D-Ala dipeptides at the ends of the muropeptides of peptidoglycan has emerged. The resistance takes the form of changing the D-Ala-D-Ala substrate to D-Ala-D-lactate which still allows the essential crosslinking of the peptidoglycan while remaining unaffected by the antibiotic. Vancomycin-resistant Enterococcus (VRE) has three different enzyme mutations to accomplish this. The first enzyme is a lactate dehydrogenase to synthesize D-lactate from pyruvate. The second synthesizes D-Ala-D-lactate from the components D-Ala and D-lactate. The third selectively cleaves D-Ala-D-Ala leaving only the D-Ala-D-lactate which is not susceptible to antibiotic action. Macrolides, streptogramins, and lincosamides can be inhibited by methylation of the A2058 adenine in the 23S rRNA. This modification prevents the antibiotic from fully binding to the ribosomal E site such that protein synthesis proceeds. Mutations in either the A or B subunit of DNA gyrase offers resistance to quinolones. Mutations in the β-subunit of RNA polymerase can offer resistance to rifampin by reducing the binding efficiency of the antibiotic to the exit of RNA polymerase such that transcription proceeds normally. Similarly a mutation in the 30S subunit of the bacterial ribosome confers resistance to streptomycin. Mutations in the enzymes which are active in tetrahydrofolate synthesis also occur and offer resistance to trimethoprim and sulfonamides. Bacteria have evolved to render antibiotics ineffective by changing the target of antibiotic activity, either the primary structure of the protein or by the addition of chemical groups.(1)

Finally, certain antibiotics require activation and resistance can be conferred by preventing this from occurring. This is true of metronidazole and isoniazid used to treat ulcers and tuberculosis respectively.(1) Given enough time, bacteria evolve mechanisms of resistance to practically any antibiotic.


http://groundupstrength.wdfiles.com/local--files/health%3Aantibiotic-resistance-questions-and-answers/antibiotic-resistance.jpg
Transmission of antibiotic resistance:

Bacteria utilize a variety of methods to propagate antibiotic resistance. Genetic transfer of resistance genes may take the form of transposons, integrons, and conjugative transposons. Transposons are pieces of DNA flanked by insertion sequences coding for the enzyme transposase that can insert themselves into the genome or plasmid of a cell. These can potentially carry, and thus spread a resistance gene. Integrons are transposons that also have an integrase gene and an attachment site. This allows circular pieces of DNA, like plasmids, to be integrated into the genome. Conjugative transposons (CTn) differ in that they transmit themselves from the genome of one cell to the genome of a second cell. The CTn excises itself, circularizes, is passed by conjugation to another cell, and integrates itself into the genome.(1)

While random mutations can and sometimes do confer resistance to an antibiotic, transmission of resistance genes already present in the population from natural selection and genetic drift is far more time and energy efficient for a bacterium. Plasmids are one mode of transmission of antibiotic resistance genes. When two cells conjugate, one which has a plasmid with an antibiotic resistance gene with a second bacteria that lacks it, the second cell gains a copy of that plasmid such that both bacteria are antibiotic resistant. It is also possible for plasmids to have two or more resistance genes. Selection for one resistance gene simultaneously selects for all the others on the same plasmid. Thus while the bacterium may not currently need these additional resistance genes, they persist in the cell and the population. Horizontal gene transfer of plasmids via conjugation which occurs even between bacteria of different species is a real threat. Combined with selective pressure from an antibiotic and short generation times, it does not take long for a high percentage of the population to become resistant to any given antibiotic.(1)

Bibliography:

(1) Wilson, Brenda A.; Salyers, Abigail A.; Whitt, Dixie D.; Winkler, Malcolm E.; Bacterial Pathogenesis A Molecular Approach. Ed. 3. ASM Press: Washington D.C., 2011. p321, 330-340, 350-367.

(2) Schaechter, Moselio; Ingraham, John L.; Neidhardt, Frederick C.; Microbe. ASM Press: Washington, D.C., 2006. p23-27, 398-99, 453-454.

(3) Kohanski, Michael A.; Dwyer, Daniel J.; Collins, James J.; "How antibiotics kill bacteria: from targets to networks." Nat Rev Microbiol. 2010 Jun; 8(6): 423–435.

OUR SOLUTION










Phage Therapy: History, Research, & Cultural Implications
Lakaysha Blacksher
Human Practices, iGEM 2015

Intro

Bacteriophage (“phage”) therapy, as a study, was initially developed in 1916 but then became nearly obsolete in the West until the 1940's and again after the fall of the Soviet Union. In the East, phage therapy had been and is still currently used in Georgia and Poland among others. Their success has thus rekindled interest and research in the West. This therapy is increasingly cited as the best alternative to antibiotics, the overuse of which has led to increased bacterial resistance.

Purpose and Function

The purpose of phage therapy, according to the Phage Therapy Center in Tbilisi, Georgia, is to provide “an effective treatment solution for patients who have bacterial infections that do not respond to conventional antibiotic therapies”. Phage therapy has been used to cure acne, some symptoms of cystic fibrosis (CF), urinary tract infections (UTIs), and countless other conditions. Bacteriophages function by infecting a bacterium, replicating inside of the bacterium, and causing the bacterium to lyse (bursting the cell membrane, spilling the cytoplasm, and killing the bacterium).

History

The first use of phage therapy is relatively unknown, given that several scientists have made the claim of discovering its usefulness. In 1896, Ernest Hankin, a British scientist, reported the presence of “marked antibacterial activity (against Vibrio cholerae), which he observed in the waters of the Ganges and Jumna rivers in India...”. In 1898, Russian bacteriologist Nikolay Gamaleya observed a similar function with Bacillus subtilis. Neither of these scientists developed their observations further. However, the British scientist Frederick Twort “reintroduced the subject almost twenty years after Hankin’s observation by...advancing the hypothesis that it may have been due to...a virus”. While Twort did not further the research due to lack of finances, a French-Canadian named Felix d’Herelle managed to further the Afterward, d’Herelle used his discovery to successfully treat dysentery in a 12-year-old boy. This became the first attempt to use bacteriophages therapeutically.

Importance

Phage therapy, while not seen as a “be-all, end-all” for infections, is offered as an alternate treatment in the fight against antibiotic resistance. Antibiotic resistance has become an ever-increasing difficulty and microbiologists have not been able to keep up with the mutations conferring resistance and the transmission of resistance genes (often occurring by horizontal DNA transfer from one resistant bacterium to a nonresistant one). One “superbug”, MRSA (Methicillin-Resistant Staphylococcus Aureus), has become common place in hospitals. While it is often not lethal, it can still pose a threat to immunocompromised patients.

Role of iGEM

This year’s iGEM team from Wisconsin Lutheran College is working with bacteriophages to combat antibiotic resistance. The TLS Phage used binds to TolC, an antibiotic efflux pump commonly found in resistant bacteria. When antibiotics are used in tandem with phage therapy, the antibiotic will eliminate the bacteria lacking a resistance gene while the TLS Phage will kill the rest.

Cultural Implications

Historically phage therapy has not been prominent in Western cultures, but it may gain popularity in the future. One must, therefore, question past and current practices: Why do we “over-medicate” with antibiotics? Is there a culture of excess? Are there consequences to overusing phage therapy as happened with antibiotics? What then?

The West is a culture of excess, and most American/European media portrays opulence as total success. In battle there is the need to annihilate the enemy in effective, though destructive ways, be it an army, a competing corporate entity, or a microscopic organism. While this phenomenon of antibiotic abuse has spread across the globe, it is not as ingrained in non-Western cultures, and moderation is still of utmost importance, especially due to cost of medical developments.

The culture of over-medicating in the US began almost as quickly as antibiotics were developed. Farmers began feeding animals antibiotics because they were often infected by bacteria present in fecal matter, near or in grazing pastures, and contaminated water. Patients in hospitals also often neglected to take their full courses of antibiotics due to miseducation and/or personal laxness. Therefore, what few bacteria survived grew resistant, so much so, that any formerly used antibiotic was rendered useless.

Should phage therapy become widely used in the West by 2020, given this culture of excess, there are concerns that this technology may be abused. Is it possible that phage therapy can be abused? For now, it does not appear so, given that antibiotics function in a very different manner than bacteriophages, are found naturally, and are essentially nontoxic, given they “consist of a core of genetic material (nucleic acid) surrounded by a protein capsid”. This is promising, especially for rural areas that may not be capable of developing new antibiotics to fight infections, and also for bacteria that simply cannot be killed by conventional means.

Advantages and Drawbacks

As with any scientific development, there are possibilities that phage therapy may not work as well as hoped or have unexpected side effects. Thus, it is always wise to review possible disadvantages and risks.

While the advantages include non-toxicity, no possibility of cross-resistance, relative cost-effectiveness, and potential for single dosing. Some disadvantages include the following:

• Phages may kill gut flora, removing any probiotics necessary for healthy digestion, and render the patient more ill.
• They also pose a danger to immunocompromised individuals by way of infection—“many protein-based pharmaceuticals can stimulate immune systems, antibiotics that lyse bacteria will release bacterial toxins in situ, and live-attenuated vaccines both actively replicate and evolve including within the context of infecting body tissues”. It should be noted that phages are viruses and should be handled accordingly.
• There is a potential for ineffectiveness if the phage lacks lytic capability (reproducing and destroying the membrane of affected cells at once) or virulence.

This may seem discouraging, but it should be noted that pharmaceutically-based phages go through a rigorous selection via the FDA, EPA, and the USDA after intense monitoring by pharmaceutical companies. They are selected by high virulence (so long as it does not subject patients to worse illness), non-toxicity, non-temperance (does not undergo lysogenesis), and other specifics.

Conclusion

It may be advisable to use phage therapy in tandem with antibiotics until the alternate becomes a necessity; however, use of pharmaceutical agents does not need to be done away with completely. The decision to use them largely rests with facilitators of medicine, such as pharmaceutical companies and hospitals, but as this technology has seen marked success in the former Soviet Union, it is increasingly evident that it is a viable weapon in the fight against antibiotic resistance. Sources Cited

Abedon, Stephen T., Loc-Carrillo, Catherine, Pros and cons of phage therapy. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278648/

Alavidze, Zemphira; Morris, J. Glenn, Jr.; Sulakvelidze, Alexander, Bacteriophage Therapy. March 2001. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC90351/ Encyclopedia Britannica, Bacteriophage.

http://www.britannica.com/science/bacteriophage

iGEM 2015, https://2015.igem.org/Team:WLC-Milwaukee/ Madhusoodanan, Jyoti, Bacteriophage Boom? 29 September 2014. http://www.the-scientist.com/?articles.view/articleNo/41097/title/Bacteriophage-Boom-/ Phage Therapy Center, About Phage Therapy Center. 2013. http://www.phagetherapycenter.com/pii/PatientServlet?command=static_about