Living Textbook MC610

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Related Issues to Antimicrobial Chemotherapy

Cell Permeability to Antimicrobial Agents:

Antimicrobial agents have to penetrate through the cell wall then the cell membrane of microorganisms. This can take place either by

  1. Passive diffusion that depends primarily on lipophilicity and is the main mode of entry into Gram-positive bacteria.
  2. Hydrophilic type interactions such as interactions with Teichoic acid in Gram-positive bacteria and with porins in Gram negative bacteria.
  3. Facilitated diffusion or active transport that may require energy and provides an efficient way for delivery of the agent into the microorganism.

In many cases the selectivity and efficiency of the antimicrobial agents are dependent on the mode of transfer into the cell.

Examples of Drug Entry into Bacterial Cells:

Tetracyclines enter against a concentration gradient. This may be due to two mechanisms; one is an active transport by an energy-requiring carrier. The second relies on the physicochemical property of the drug and the bacterial cell. Tetracyclines are poly-charged molecules. At physiological pH about 7% of the agent is uncharged. The bacterial cell maintains a slightly higher pH inside the cytoplasmic membrane, so after the uncharged Tetracycline enters the cell, most of it becomes charged, allowing more Tetracycline to diffuse into the cell as uncharged species to achieve equilibrium. These methods of entry are the basis for the selectivity of Tetracyclines to bacterial cells.

Flouroquinolones may enter by simple diffusion of the uncharged species (around 10% of the drug at physiological pH), but mainly enter into bacterial cells by chelating with Magnesium in porins and passing through. When it reaches the periplasm, the chelate dissociates due to a drop in pH releasing the free drug that passes into the cytoplasmic membrane. This explains why some of the Quinolones are more effective against Gram-negative bacteria.

Intrinsic Resistance to Antimicrobial Agents:

Pseudomonas aeruginosa, the dangerous opportunistic Gram negative bacteria possess a low efficiency porin that restricts diffusion to about 1% of the rate of other Gram negative bacteria. This property of the porin channel coupled with the rigid outer membrane are the main reasons for the intrinsic resistance of this species to antimicrobial agents.

But the very low permeability of the outer membrane to compounds cannot by itself account for the exceptional resistance of this strain to antimicrobial agent. Another important factor is the presence of an efflux system in these bacteria that can extrude different agents including in case of pseudomonas Tetracyclines, Chloramphenicol and some Quinolones. These efflux systems are in the form of proteins embedded in the cytoplasmic membrane and outer membrane. It is not exactly understood how the efflux systems work, but it seems that they coordinate to form a channel in the outer membrane that transports the drugs to the outside of the cells, and that this process requires energy.

It is important to note that the efflux system has to work in synergism with low permeability to the drugs. If the outer membrane is highly permeable to a drug, the efflux system will be overwhelmed with the drug molecules, and will not function properly and the intrinsic resistance is lost.

These efflux systems are present in other bacteria such as E. coli and in many cases cause multidrug efflux.

Acquired Resistance to Antimicrobial Agents:

The first reported incidence of resistance dates back to 1907 when Paul Ehrlich encountered resistance against arsenic chemotherapy. But this problem exploded with the introduction of Sulfonamides and antibiotics into clinical setting. In early 1940 when Penicillins were first introduced, less than 1% of Staphylococcus aureus showed resistance. This number rose to 14% by 1946, 38% the following year and today has reached 90%. Resistance has now spread from antibacterial and antimalarial drugs to antifungal and antiviral agents as well.

A good example of how the use of an antibiotic can affect emerging resistance is Vancomycin. It has been used for over 30 years, and had not shown resistance, due to infrequent prescription of the drug since its early preparations showed high level of toxicity. With better preparations that show lower toxicity and the emergence of resistance strains of bacteria that responded only to Vancomycin, its clinical use increased and resistance started to emerge.

Factors that appear to propagate resistance include wide availability of antimicrobial agents, irrational use and abuse of these agents, use in animal husbandries, especially as growth promoters and the wide use in lotions, soaps and other household items.

Genetic Basis for Bacterial Resistance:

The first question that comes to mind is where did the resistance genes initially come from? The most logical answer is that they are produced by the bacterial species that produce the antibiotic to protect them against the action of that agent. With time, other bacteria developed these genes either by simple mutations or through gene transfer to protect them from specific antimicrobial agents. They start as a few organisms in the population, but after the introduction of the antibiotic it kills the sensitive bacteria, leading to an increase in the resistant type and a shift in the population from the sensitive to the resistance type.

Although many of these genes have evolved hundred of million years ago, there is evidence for the evolution of some modes of resistance developed during the modern era of chemotherapy. Simple mutations have been reported to give rise to resistant bacteria during anti-TB treatment. However, simple mutations are rare and cannot account for the wide spread of resistance that is seen today. Several processes have been shown to propagate bacterial resistance:

  1. Gene Transformation. Bacteria can be transformed by naked DNA released from lysed bacterial cells that are absorbed into another bacterial cell. It is then integrated into a homologous region on the recipient genome. An example of this is emergence of mosaic genes that arise by interspecies genetic recombination.

Resistance to ß-lactam may arise due to mutations to PBP. This is remarkable, since ß-lactam can bind to different types of PBP, so any resistance will have to involve mutations to all types of PBPs. How can this happen?
There is evidence now that recombination among PBP genes from different genes is the major cause of low-affinity PBP seen in these resistant bacteria. This results in a mosaic structure and is believed to be the case in resistant Neisseria sp.

  1. Transposons . While the same low affinity PBPs mentioned earlier are seen in MRSA it has been concluded that this organism cannot undergo transformation as easily and that the spread of such a gene is through transposons. Transposons are hopping genes that are present in many Gram positive and negative bacteria and can integrate themselves into plasmids and chromosomal DNA in recipient cells even if there is no homology to the sequence.
  2. Plasmids . In Gram-negative bacteria, the most significant mode of spread of resistance is through R-plasmids. They are DNA that are separate from chromosomal DNA and its spread occurs through conjugation between an R+ and an R- bacterial cell. The wide spread of R-plasmids seen today is due to the widespread use and abuse of antimicrobial agents, with animal use playing a significant role.
  3. Bacteriophages. They are viruses that invade bacterial cell, integrate their own DNA into the bacterial chromosome and replicate forming new phages. During its release from the chromosome, it may pick up a resistant gene and transfer it to other cells that it invades.
  4. Gene Regulation . Regulators of several resistant genes on the bacterial chromosomes are only activated by the presence of the antibiotics. For examples the efflux system along with reduced permeability of several antimicrobial agents is activated by the presence of only one of these agents, leading to a multidrug resistance species.