Antibiotic Resistance

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)

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)

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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.

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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.

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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.