Antibiotic Resistance, Superbugs and Drugs

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Antibiotic resistance results, because spontaneous mutations occur so frequently that all bacteria are different.  It is just a matter of exposing enough bacteria to an antibiotic to find one that is insensitive to a particular antibiotic.  More bacteria mean a greater chance of mutations to antibiotic resistance.  The gut contains a lot of bacteria and sewage treatment plants are loaded with gut flora.

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Antibiotics are Ubiquitous

All organisms, plants, fungi and animals/humans produce chemicals that kill bacteria, i.e. antibiotics.  I have written many articles about the natural antibiotics of plants, a.k.a. phytoalexins or “antioxidant” polyphenolics, and the human defensins that are peptides with heparin binding domains.  Bacteria also produce viruses, called bacteriophages, that kill other bacteria.  All of these natural antibiotics are small molecules that interact with many different human proteins, and it is these side effects that permit their exploitation as pharmaceuticals.  Thus, statins were selected from fungal antibiotics that inhibited an enzyme needed for human synthesis of cholesterol, metformin was a phytoalexin found to reduce blood sugar and resveratrol is a grape phytoalexin.

Plant Antibiotics are Natural

The flavoring chemicals in herbs and spices have a far more important use in food preparation than titillation of taste buds, since those chemicals kill common food pathogens.  More profoundly, it is important to realize that the selective advantage of phytochemicals/polyphenols/alkaloids/essential oils to the plants that make them, is as natural antibiotics.  Plants kill bacteria, as well as fungi and insects, for a living.

Plant Chemicals Attack all Aspects of Bacteria

Most of the thousand genes that are present in a bacterium code for proteins/enzymes and most antibiotics target those enzymes.  Penicillin binds to an enzyme needed to make bacterial cell walls, streptomycin target protein synthesis, rifampicin blocks RNA synthesis, actinomycin D inhibits DNA synthesis, etc.

Mutation to Antibiotic Resistance is Automatic in Bacteria

Each time a cell replicates, mistakes are made and the new DNA molecule of each chromosome is slightly different than the original.  There are about a thousand genes on the single chromosome of a bacterium and about the same number on each of the 23 human chromosomes.  About a dozen mistakes, mutations, are made each time bacteria replicate.  The mutations that alter the gene target of an antibiotic and produce a bacterial enzyme that is unaffected by the antibiotic, yield an antibiotic resistant bacterium.  The mutant gene now codes for antibiotic resistance and the presence of several resistance genes in the same bacterium produces multiple antibiotic resistant "superbugs."

Mutations are Random, but Antibiotics Select for Resistance

Each cellular replication produces random mutations throughout the bacterial DNA, but of the billion sites along the DNA that can mutate, only a few will produce a modified enzyme that will no longer interact with a particular antibiotic and thus be resistant.  Antibiotic resistance mutants are rare, less than one in a million, but a million bacteria can grow from a single cell in a day and occupy a volume less than a crystal of salt.  Ten hours later, after ten more doublings of the million bacteria, there will be a billion, and there will be a good chance that among those will be a mutant that is resistant to a particular antibiotic.  In the pound of bacteria in the human gut, there are mutants that are resistant to most antibiotics, including the antibiotics that have not yet been developed.  Of course, most of those antibiotic resistant bacteria are just flushed down the toilet.  Treatment with antibiotics kills all of the sensitive bacteria and leaves only the resistant.  Thus, antibiotic treatments select for antibiotic resistant bacteria.

Common Use of Antibiotics Selects for Resistance on Plasmids

Genes are transferred between bacteria by bacteriophages, conjugation (a kind of bacterial sex) and transformation, which is the release of DNA from one bacterium with subsequent uptake by another.  Biofilms, which are communities of many different species of bacteria, stimulate transformation and exploit bacterial DNA as a matrix material to hold the communities together.  The human gut is lined with biofilms and the biofilm bacteria secrete vitamins as the quorum sensing signals that coordinate community activity.  Thus, some vitamins must stimulate transformation, the exchange of DNA among members of the different species of bacteria in the biofilms with evolution of new and novel species.  Rapid change in the gut environment selects for a shift in genes that provide for adaptation to the new environment to small DNA fragments, plasmids, that move most readily between bacteria.  Antibiotic treatment results in antibiotic resistance genes on plasmids.

Use of Multiple Antibiotics Selects for Multiple Antibiotic Resistance Plasmids

Persistent use of an antibiotic will spread resistance to a particular antibiotic through the gut flora, facilitated by antibiotic resistant plasmids.  Replacement of a second antibiotic will result in a new plasmid with both antibiotic resistance genes.  Hospitalization and exposure to a plethora of bacteria with multiple antibiotic resistance plasmids will result in rapid conversion of gut flora to multiple antibiotic resistance upon exposure to any antibiotics.  Hospital staff would be expected to be natural repositories for multiple resistance genes, especially if they are exposed to any antibiotic (or pharmaceutical.)

Most Pharmaceuticals Select for Multiple Antibiotic Resistance Plasmids and Superbugs

The frightening rise of superbugs resistant to all known antibiotics has been attributed to the accelerated use of antibiotics in medicine and agriculture.  Mixing megatons of bacteria in the guts of billions of people with tons of antibiotics, and still more in sewage treatment plants and agriculture, is bound to produce bacteria with every type of multiple antibiotic resistance plasmid imaginable.  But that is not the biggest problem, since fingering the commercial use and misuse of antibiotics ignores biggest exposure of bacteria to antibiotics.  It ignores the fact that most popular pharmaceuticals, NSAIDs, statins, anti-depressants, anti-diabetics, etc., also have substantial antibiotic activity.  Most of these pharmaceuticals started out as phytoalexins and then were found to also have pharmaceutical activity.  Pharmaceuticals are just repurposed natural antibiotics.  When you take an aspirin or Metformin or a statin, you are taking an antibiotic.  When you take a pharmaceutical, you are selecting for multiple antibiotic resistance plasmids in your gut flora and you may be making the next superbug.

Health and Heparan Sulfate Circulation — Connective Tissue is Alive

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Arthritis, Alzheimer’s, diabetes, cardiovascular disease, osteoporosis, cancer, etc. are all diseases of cellular metabolism and secretion.  What goes on inside cells and on their surfaces explains a lot about health and why we get sick.  Cells feed off of what’s around them, use some of those materials to replicate and package up cell-made materials for export.  Eat, replicate and secrete.  Symptoms of disease result if those processes are compromised.

Cell that make Cartilage, Eat Cartilage

The connective tissue that makes up the cartilage of tendons and the non-mineral parts of bones, as well as a layers of skin, is made up of proteins (collagen) and polysaccharides (glycosaminoglycans, GAGs), e.g. heparan sulfate, hyaluronan and chondroitin sulfate, produced by  chondrocytes or fibroblasts.  These proteins and polysaccharides are synthesized and then secreted by cells.  This process goes on continuously, since the connective tissue is alive and literally crawling with cells that make the cartilage.  To keep the connective tissue healthy, the old tissue has to be digested, so that new material can replace it.  Thus, the cells that live in cartilage also eat cartilage.  These cells get all of their nutrients, e.g. protein and carbs, from eating cartilage.  They don’t get glucose and amino acids, or even oxygen (they ferment), from the blood, because there are no blood vessels in cartilage.  The photomicrograph at left shows the red chondrocytes surrounded by a light capsule of heparan sulfate as they burrow through the purple cartilage.  The next micrograph shows the cytoskeleton of actin filaments (stained with a red fluorescent dye, that lies under the cytoplasm of a chondrocyte.  Motor proteins move other proteins, such as syndecans, the proteins to which the heparan sulfate chains are attached, through the  cell membrane (see the animations below.)  The last micrograph shows the green stained microtubule network on which vesicles move to carry heparan sulfate products from one end of the cell to the other (under the actin and past the orange-dyed nucleus) during synthesis and digestion.

Chondrocytes Burrow Through Cartilage

Chondrocytes are the cells that eat and make cartilage, but all of this eating and making goes on at the same time that the cartilage is also holding everything together, i.e. it is still strong.  If cartilage is cut and the cut ends are held tightly together, the chondrocytes will knit the cartilage together and it will become as strong as it was. 

Heparan Sulfate Circulates over the Surface of Cells

Chondrocytes are not actually rigidly embedded in the cartilage, but rather maintain a capsule of heparan sulfate around themselves.  Thus, they continue to secrete a mixture of heparan sulfate, chondroitin sulfate and collagen, but the heparan sulfate is recycled through the capsule and the other molecules merge into the existing cartilage.  Thus, the heparan sulfate is a kind of carrier that keeps the cartilage from “setting up” while it is being made and transported.  Other cells of the body, such as neurons, don’t make cartilage, but they still have heparan sulfate (HS) circulation that is intimately involved in many other processes, such as the action of hormones.  Disruption of HS circulation causes the symptoms of Alzheimer’s or type 1 diabetes, for example, since amyloids assemble as filaments on threads of HS, and the amyloid filaments jam essential HS circulation.  Plaque in atherosclerotic vessels is high in HS content.  HS is also a major component surrounding vessels to form the blood brain barrier and the barrier to protein loss from kidneys into urine or loss into the gut lumin.  Heparin (fragments of HS) is continually released from mast cells in the lining of the gut to prevent pathogens from binding to cell HSPGs. 

HS Sweep the Cell Surface

There is a constant flow of heparan sulfate proteoglycans (HSPGs) through the cell membrane from the rear of the chondrocyte to the front where the HS is digested again and the protein that was embedded in the membrane, syndecan, is recycled to the Golgi for another trip.  HSPGs (animation to left with blue protein and yellow HS) are attached to motor proteins that propel them through the membrane along microfilaments of actin that form the cyctoskeleton just under the membrane in the cortical region of the cell.  Thus, the heparan sulfate of the HSPGs stick out like hair from the cell surface and sweep continuously from the back to the front of the cell.  At the front of the cell, the HS sweeps through the intact cartilage and reverses the process of cartilage assembly.  The chondroitin sulfate, collagen and HSPGs are dragged into the cell and digested.  The protein parts of the HSPGs are transported to the Golgi  and the HS is synthesized along with other cartilage components and moved in vesicles along microtubules before it is secreted.

HS is Secreted at One End and Eaten at the Other

The animation left shows 1) the initial digestion of the cartilage proteins and polysaccharides on the left.  These cartilage components of amino acids and sugars, are used by the chondrocytes as their sole nutrients 2), and to produce new proteoglycans 3) HS and chondroitin sulfate proteoglycans, in the Golgi, are 4) packaged into secretory vesicles and are 5) secreted on the right.  The HS chains, attached to proteins, are 6) swept through the membrane (see the first animation above) toward the front of the cell, leaving the collagen and chondroitin sulfate for form cartilage behind.  In the process, the heparan sulfate proteoglycans 7) disrupt and solublilize old cartilage ahead as the chondrocytes 8) move through the connective tissue like moles digging through soil.

Other Cell Processes Involving Heparan Sulfate:

• Amyloids of Alzheimer’s and type I diabetes assemble bound to HS.
• Hormones bind to receptors wrapped around HS.
• Blood clotting is controlled by HS.
• Complement is controlled by HS.
• Blood brain barrier is composed of HS.
• Kidney protein barrier is composed of HS.
• Inflammation blocks HS synthesis and promotes heparanase synthesis.
• GAGs are animal soluble fiber when eaten and feed gut flora.
• Pathogens bind to HS.
• HIV-TAT is transported between cells by HS circulation.
• Heparin is made by heparanase fragmentation of HSPG in mast cells and is secreted along with histamine. 
• NFkB activation inhibits HSPG production and stimulates heparanase production.
• Heparan sulfate proteoglycans organize nerve synapses and acetylcholine esterase binds to HS. 
• Gastric proteases cleave around heparin binding domains of proteins, e.g. milk, consist of clusters of basic amino acids.  Peptides with heparin binding domain are antimicrobial; all of the heparin binding peptides are subsequently degraded by pancreatic proteases.
• Heparanase is initially secreted inactive and bound to HSPGs, but it remains bound and is internalized again along with the recycling HSPGs, and is activated before being secreted again.
• Allergens and autoantigens are unusual proteins with sequences of three adjacent basic amino acids (arginine or lysine) that require HSPG circulation for presentation of the immune system.  Nuclear proteins that interact with nucleic acids have sequences of four basic amino acids, the nuclear translocation signal, and are therefore common antinuclear auto antigens.

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