Structural Biochemistry/Drug Resistance

Antimicrobial resistance – also known as drug resistance – occurs when microorganisms such as bacteria, viruses, fungi and parasites change in ways that render the medications used to cure the infections they cause ineffective. When the microorganisms become resistant to most antimicrobials they are often referred to as “superbugs”. This is a major concern because a resistant infection may kill, can spread to others, and imposes huge costs to individuals and society.

Due to the amount of antibiotics currently being used for humans and agriculture, there has been an increased amount of drug resistant pathogenic bacteria. This resistance is a result of natural selection, in which the pathogens resistant to the antibiotics survive and continue reproducing, and the nonresistant strains are killed.

Introduction
When penicillin was discovered in 1928, the effectiveness and convenience of antibiotics for treating many infectious diseases was realized, and taken for granted. Now, the use of antibiotics has become increasingly commonplace, and thus allowed for the development of more drug resistant strains of bacteria.

These bacteria that have grown to become more resistant to the past drugs lead to new generations of medication that are able to deal with these infectious diseases. The only problem that is realized that many of these drugs will become useless because of the mechanisms that the bacteria display in where they will evolve into better bacteria then they were in the past. Bacteria, who have similar structures and genes, are able to evolve through the accumulation of multiple genes that code for drug resistance, pumps that work in 3 different areas of the bacteria, and the similarity of these drugs that are already in bacteria which help them become resistant. (Nikaido 2011)

Mechanisms of Drug Resistance
There are many reasons as to why certain bacteria strains can become resistant to certain drugs. Most of these resistances originate from the bacteria that were used to produce the antibiotic, because they need to be resistant to their own product, or from microorganisms that exist in the environment and thus are exposed to the excess amount of antibiotics used today.

Changes in the target protein
Bacterial genes may mutate and affect the protein that the drug targets. Due to this change, the protein may be less susceptible to the biochemical effects of the drug. While these mutations may not be carried on to further generations under normal situations, the presence of the antibiotic drugs will affect the selection process, favoring only the bacteria with this mutated protein form. Resistance of this kind is affective against man-made drugs that cannot be inactivated enzymatically.

Inactivation through enzymes
Certain drugs are more susceptible to being inactivated by enzymes that are produced by certain bacteria. The genes that code for these enzymes usually originate from the bacteria that were originally used to produce these antibiotics. Examples of enzymatic drug deactivation can be seen in the phosphorylation/acetylation/adenylation of aminoglycosides and the hydrolysis of β-lactamases.

Aminoglycosides are inactivated by the reducing net positive charges on these antibiotics. These modifing enzymes, such as AAC(3)-11, will act on the position 3 of the substrate that belong to the phylogenic group among the enzymes. Due the antibiotic-producing micro-organisms that are found in these bacteria, they are already present amongst their DNA which make them resistant to these aminoglycosides. (Nikaido 2011)

B-Lactam resistance was caused by the B-lactamase coded in by plasmid genes. This was problematic because they are resistant to many drugs like methicillin and similar compounds that are able to be hydrolyzed through different enzymes like Tem B-lactamase & AmpC. These drugs were recreated for a 2nd generation & 3rd generation, but they the AmpC enzyme was able to evolve also to counteract these drugs. Soon after, older versions of drugs would have to be introduced to counter the enzymes like Vancomycin, which binds itself to a substrate that is a precurse to the cell wall peptidoglycan instead of inhibiting the enzyme itself. (Nikaido 2011)

Acquisition of other genes
The sequencing of genes for penicillin resistant Streptococcus pneumoniae showed that the target proteins were being produced as mosaic proteins (parts came from other organisms). The drug was ineffective due to the change in the targets of the antibiotic.

R plasmids are usually transferred very efficiently in cell to Cell transfers which have been made possible between the bacterial cells because they are closely related to each other. R plasmids, which are highly stable, also helps their case in their increasing drug resistance. The R plasmids, containing drug resistance genes, are able to deliver these genes to any piece of DNA because they are composed of transposons. These R plasmids contain a unique 59-base-3'-sequence called an integron which catalyzes an insertion of resistance genes into a compatible site. Through this process, more drug resistant genes are able to be passed to multiple bacteria for a higher drug resistance. (Nikaido 2011) (Kaiser 2011)

Host cells often lose R plasmids acquired from cloning vectors in large numbers. However, most naturally derived R plasmids are stable and lost less often when the new host cells are multiplied. This can be attributed to the fact that natural plasmids have in their structure "killer" elements that kill the host cell when plasmids are lost. (Nikaido 2011)

In addition, genes can acquire drug resistance through mutations, extrachromosomal DNA transfer, such as conjugation, which is the transfer of the R-factor plasmid via pilli, transformation by obtaining DNA from the environment, and transduction via the R-factor on the bacteriophage. Also, genes can become resistant to drugs through transposable drug resistance sequences using transposons, or small DNA segments that move from one DNA molecule to another.

Preventing drug access


A drug can generally be caused to be less effective if its access to its target is limited. Locally, a bacteria can produce certain proteins that will effect conformations of ribosomes or DNA and thus restrict the access of the antibiotic to those target areas. Another method of this mechanism is the use of drug specific efflux pumps.

Reducing the access of drugs inside the bacteria have induced a nonspecific inhibition. This inhibition is created where bacteria have decreased their outer-membrane permeability in which it reduces the access of drugs entering the bacteria. Often, porin-deficient mutants are selected. Being a double edged sword, the lower permeability of the outer-membrane will also reduce the intake of nutrients entering the bacteria thus being detrimental to cell. Mutations in the coding sequences of the porin have been discovered that lower the permeation rate of bulky antibiotics but leaving small nutrient molecules unaffected, allowing them to permeate at normal levels.(Nikaido 2011)

The next method is through multidrug efflux pumps that were first identified in E. coli & P. areuginosa. These pumps have been discovered in most clinical gram-negative bacteria that share similar systems with P. areuginosa. The Multidrug Efflux Pumps are consisted of 3 different parts where it has a resistance nodulation division exporter protein, a gated outer membrane, and a membrane fusion protein. (Nikaido 2011) (Aeschlimann 2011)

The Major Facilitator Superfamily are the largest families of transporters and contain many efflux pumps. In this class, QacA & QacB are the first examples of pumps in which the QacR-inducer complex has been able to accommodate diverse ligands with their binding site. They will be able to bind to these drugs that have entered through the membrane and then be pumped out of the system. Some of these efflux pumps will always be pumping in which a repressor is needed in order to stop the operon from letting the pumps continue their function. (Nikaido 2011) (Aeschlimann 2011)

The Small Multidrug Resistance family have different efflux pumps that were coded and to be seen on the chromosomes of gram-negative bacteria. The trasnporters of these pumps will encounter substrates in which they will be deprotonated and be pumped out by the inward flux of protons. (Nikaido 2011)

The next class of efflux pumps belong to the Resistance-Nodulation-Division Family in which these pumps are associated with 2 other classes of proteins, the outer-membrane channel and membrane fusion protein. The construction of this pump gives a huge advantage to the bacteria because it gives a direct export of these drugs into the medium and outside of the bacteria where they will have to re-enter the bacteria through the outer membrane barrier.Nikaido 2011)

These pumps will work all together creating synergy in which they will pump out most antibiotics and becoming drug resistant. The more pumps there are, the more drug resistant it will become. Although some pumps cannot carry out some antibiotics, Homologs of AcrD can carry out this function in which they will make bacteria even more resistant to antimicrobial agents. (Nikaido 2011)

Persister Cells
When high concentrations of antibiotics are introduced to the bacteria, it would be safe to assume that all bacteria will be killed. But a special phenomenon happens when some of the bacterial cells will survive the antibiotics. These cells are called persister cells in which has become a strategy for bacteria to generate drug-resistant populations. Bacteria produce phenotypically different mixtures in populations so that any one of them can be beneficial in changing environmental factors. Thus, antibiotic therapy is deemed inefficient in the presence of such persisters.

These cells are similar to spores in which small portions of these cells are dormant in the bacterial population. Being dormant, they will not react to the antibiotics making them possible to reoccur the infections once the antibiotics are gone. There are no current treatments for persister cells, but there possible ways to target the cells through anti-microbial peptides. These AMP will target dormant or active cells which will be effective in attacking persister cells. (Nikaido 2011) (Duchene 2011)