by Daniel Keays

          Antibiotics aren’t new. Microbes have been producing them for millions of years to help themselves gain an ecological advantage. And antibiotic resistance isn’t new either. The target microbes have been producing the means to avoid destruction by the chemical substances directed against them for just as long. Humans just happened to discover this world of microbial biochemical warfare in the 1940s. It’s still a little new to us, but the biological processes have been firmly developed.  

          Because of the variability of microbes, antibiotics don’t work against all bacteria all the time. The number of different types of bacteria susceptible to a given agent is called the spectrum:  An antibiotic that can kill a wide range of organisms is called a broad spectrum; one whose range of activity is confined to a few species is called a narrow spectrum. Choosing a proper antibiotic to treat an infection often depends on knowing which microbe is causing a disease. When known, it is best to use a narrow-spectrum drug to prevent collateral damage to our helpful microbes. A broad-spectrum drug is usually used if the infectious agent is not known, as is often the case early in an infection before laboratory tests are available. Sometimes, when the infection is serious, and the infectious organism isn’t known, it is necessary to administer an “antibiotic cocktail,” several broad-spectrum drugs at once, hoping that at least one will do the job. When laboratory results are known, then a more appropriate choice is made.

          Antibiotics are poisons. They are designed by nature to kill. Fortunately for us, bacteria possess structures and enzymes different from ours, and they are the targets of antibiotics.  Substances like bleach kill bacteria, but clearly, giving an intravenous administration of bleach is a very bad idea. We want something that will kill bacteria but not harm us too much. Antibiotics meet the need.

          When Ernst Chain worked on penicillin in the early 1940s, he noticed that some bacteria, mostly Gram-negative rods, were naturally resistant to it.  But he also noticed that a few strains of Staphylococcus, which were on initial investigation susceptible, had become resistant to the drug.  The antibiotic resistance mechanisms bacteria had developed in the natural setting eons before were fully operational when antibiotics were introduced. It didn’t take long before the phenomenon of antibiotic resistance was well-known. Today over 90% of Staphylococcus aureus causing infections are resistant to penicillin.

            The genes that code for the resistance proteins can be passed between microbes by several means, giving rise to clones of resistant bacteria.  Because of the widespread use of antibiotics in healthcare, these genetic resistance exchanges play out there on a dynamic scale.  The more antibiotics are used, the more resistance mechanisms are pushed.  When exposed to an antibiotic, resistant bacteria survive, passing on their resistance genes—simple survival of the fittest.

 

          Bacterial genetics starts with its genome, the long strand of DNA that encodes most of the organism’s genes. It’s often referred to as a “chromosome” because its function is the same as that for higher animals, the coding of genetic information. But it isn’t a true chromosome like ours. Instead, bacterial DNA is a long strand of the classic double helix, tightly coiled and rambling throughout the bacterial cell. Bacteria don’t have a nuclear membrane to contain it.  

          In addition to the long, coiled strand of DNA we sometimes call a chromosome, bacteria often have additional, much shorter, independent strands of DNA called plasmids. Bacterial cells may contain one or several. Although they are not nearly as big as the main chromosome, they code for proteins just the same, and they usually divide in harmony with the rest of the cell.  

          The plasmids harbor information that allows the organism to adapt better to its environment. They are best known for containing genes whose products enable the microorganisms to produce enzymes that combat antibiotics, either destroying the antibiotic or altering its binding site, rendering it ineffective. Plasmids do other things, of course, such as enabling the organism to take advantage of an environmental change, such as acquiring a nutrient.

            As with any creature subject to the biological laws of natural selection, the bacteria which can rapidly adapt to changes in their environment will be the most likely to survive and pass on their advantageous genetic information to their progeny. Members of the Enterobacteriaceae are very good at this. They reproduce by binary fission, so mixing genes, as in vertebrates, is impossible, but they have several methods of exchanging genetic information.  

            One means of gene transfer is bacterial “sex,” or conjugation.  On the periphery of each Gram-negative cell are appendages called pili (or fimbriae), tiny hair-like structures commonly used by the organism to adhere to a cellular surface. Some of these become specialized to serve as a tube through which genetic material, mainly a plasmid, can travel from one organism to another.  One organism “docks” with another, the specialized pilus is hooked up to the adjacent cell, and the plasmid transfer occurs with the genetic material flowing right through the pilus tunnel.  

            Many people don’t realize it, but a virus can infect a bacterium. It’s very common. The virus that infects a bacterium is called a phage, and there are lots of them. Quite a few phages integrate themselves onto the bacterial chromosome. When they leave, they can take with them small bits of bacterial DNA and transfer them to the bacterium they next infect. It’s a way in which chromosomal DNA is transferred between organisms. It’s called transduction.

            As we all know, James Watson and Francis Crick reported that DNA strands contain the genetic code. That was in 1951. But Watson and Crick didn’t operate in a vacuum. Some twenty-three years before them, in 1928, British bacteriologist Frederick Griffith made a discovery that “transformed” the biological world. In Griffith’s experiment he used two strains of the bacteria known as pneumococcus, a species of Streptococcus that is sometimes highly pathogenic due to a slimy capsule surrounding the cell, aiding it in evading white blood cells. Of Griffith’s two strains, one contained a polysaccharide capsule enabling it to cause disease leading to the death of mice when injected. The other was a non-virulent strain that lacked a capsule. When injected into mice, the encapsulated strain rapidly killed the mice. The non-capsulated strain does nothing; the mouse immune system gobbles it up.  

          Griffith took some encapsulated deadly bacteria and killed them, rendering them, of course, non-infective. He injected them into mice along with some live non-virulent bugs. One would think that nothing would happen to the mice since the only living strain injected was the non-capsulated, nonvirulent type. But one would be wrong. What Griffith found eventually changed the course of biology, although it went virtually unnoticed at the time. The non-virulent strain became virulent by acquiring a capsule from the dead organisms, killing the mice. It was transformed. It took some dozen or more years for anyone to figure out why, but Oswald Avery and his co-workers at the Rockefeller Institute in New York figured out that the transforming factor was DNA. The non-capsulated pneumococci picked up some DNA from the dead virulent bacteria and incorporated it into their genome. Knowing that DNA was the molecule responsible eventually led Watson and Crick to do their work explaining the DNA double helix. 
          Frederick Griffith was killed in a bombing raid in London in 1941, and his discovery received little notice during his lifetime. Still, his research laid the groundwork for some of the most important discoveries in biology.
          Many bacteria can retrieve DNA strands deposited in their environment on the death and lysis of other organisms, and integrate them into their own genome. The term used for it harkens back to the one used by Griffith, transformation. Not only can an organism capture DNA from other members of the same species, but it can also get it from other genera. DNA transformation is a potent tool bacteria use to pass on genetic information.

            Another genetic discovery of great importance in the pre-Watson and Crick era was made by a botanist who specialized in genetic studies, Barbara McClintock. She was unique in two ways. She was one of the few elite women scientists of her era, and one of the few scientists interested in and working on genetics. Her discovery came using the corn plant Zea mays, commonly called maize. This variety of corn plants has multicolored kernels and lends itself well to the application of Mendelian genetics. Dr. McClintock described what is commonly called “jumping genes,” or genes that are movable from one part of the chromosome to another. Like Griffith’s discovery, her work received very little notice at the time of her discovery in 1941, but as time went on, the profound nature of the discovery was more appreciated. She received the Nobel Prize in physiology in 1983, over 40 years after her work was published.
          Jumping genes are technically called transposable elements, or TEs. “Jumping genes” is much more fun to say, but they are now usually referred to as transposons. They exist not only in plants but are very common in bacteria. By having its genes move around and not be held in a fixed location, the encoded information has much more of a chance to be displayed, copied, and actualized. Transposons give the organism much more flexibility in adapting to environmental changes.  

            One of the most significant early discoveries of molecular genetics was made by a French biologist and doctor who was nearly not around to uncover it. Francois Jacob was born in Nancy, France, in 1920. A devoted scientist, he trained to be a doctor and wanted to become a surgeon, but he faced a formidable obstacle: World War II. After joining the army, he fled France for England when the Germans overwhelmed his country. He served in the Allied military as a doctor and saw considerable action. Severely wounded at Dunkirk, he nearly lost his life. Dr. Jacob was hospitalized for seven months and survived but could never become a surgeon because of his extensive wounds. Still, he was determined to carry on work, in his words, “into the nature of things,” and ended up as a bacteriologist at the Pasteur Institute. There, he teamed up with Jacques Monod to make one of the most critical molecular genetic discoveries of all time: the lac-operon.  
            Until the work of Jacob and Monod, it was assumed that organisms cranked out the proteins and enzymes encoded in their genes all the time, whether they needed them or not. If there is a gene on the chromosome for something, it gets transcribed into messenger RNA like all the others, and that’s that. But Jacob had noticed that E. coli didn’t always make the enzymes necessary to ferment lactose, the sugar found in milk. If much glucose is present or lactose is missing, the enzyme that breaks down lactose, known as galactopyranosidase, is not made, nor are any other enzymes needed to bring lactose into the cell. This puzzled him greatly until he had an “Aha!” moment while sitting in a movie theatre with his wife. He reasoned that the genes coding for lactose fermentation were covered up and not copied until lactose was present in the environment and glucose was missing. The covering-up molecules were called operons, and since the first one described was for lactose fermentation, it was abbreviated to “lac-operon.” Jacob and Monod were awarded the Nobel Prize in Physiology or Medicine in 1965 for their discovery.
            We now know that this shielding of specific genes on the DNA strand doesn’t apply just to bacteria. The phenomenon is widespread and plays a major role in the expression and repression of the cells of our immune system. Not expressing and copying genes whose products would have no immediate role is a very efficient way to conserve precious energy. It gives those organisms that employ it a great ecologic advantage.
          Some bacteria have the coding on their DNA for enzymes that can inactivate an antibiotic. But if the antibiotic is not present in its environment the genes for the enzyme are suppressed. Only exposing the bug to the antibiotic can unlock the genes.

           A spontaneous mutation is still another way bacteria can adapt to environmental changes. With the nucleotides of DNA being transcribed by RNA polymerase billions of times a day, there are sure to be a few errors that occur. Most of the mistakes result in minimal alteration in the protein produced. A few can be detrimental to the organism. But now and then, changing just one amino acid of a protein can make a profound difference in the way the protein operates, giving the altered organism an ecological advantage. 

            In summary, bacteria have multiple ways they can alter and manipulate their genetic foundation to survive better and take advantage of changes in their environment.  A single gene may become slightly altered, giving rise to a modified protein (mutation). Bacteria can absorb and incorporate into their own genome the genetic material from another bacterium (transformation). Genetic material can be passed from one organism to another through a specialized pilus (conjugation). Genetic material can be carried from one strain of bacteria to another by a virus (transduction). Genes can move around on the chromosome and plasmids to be more accessible (transposons). Plasmids are little DNA fragments that code for proteins independent of the main bacterial chromosome. They similarly can be transferred from one organism to another. And, of course, much of the genetic material is covered and controlled by operons, enabling the organism to display “just in time” efficiency.

            From an infectious disease perspective, we think of two important outcomes that result from this genetic adaptability. One is the ability to acquire and employ virulence factors making the organism a more potent pathogen. The other is the capacity to become resistant to antibiotics and other antimicrobial agents.

            Over the two decades following the work on penicillin, new antibiotics were discovered, tested, and released regularly. One could call the 1950s and 60s the “golden age” of antibiotic discovery. Not only were molds found to produce them, but soil bacteria also. Drugs were assigned to a “class” based on their mode of action and chemical composition. Pharmaceutical firms chemically modified them to gain a broader spectrum or more convenient administration means. The supply of new classes of antibiotics seemed limitless.

            Until it wasn’t. By the early 1970s, the discovery of new antibiotics produced by molds or soil bacteria came to a screeching halt. New antibiotics were tested and released, but they were modifications of existing drugs. Several new classes of drugs were introduced in the 1990s, such as fluoroquinolones and linezolid, but they were not produced by microbes but by chemists in a lab. The dried-up pipeline for new antibiotics wasn’t for lack of trying. Thousands of soil samples and fungi from around the world were sent to pharmaceutical companies by their employees and other connections. They were meticulously screened for any hint of an antibiotic producer. Alas, nothing significant was uncovered.

            Pharmaceutical firms today still release new antibiotics, but they are invariably a “variation on a theme:” Taking a known antibiotic and modifying its chemical structure to create a drug that microbes haven’t encountered before, so resistance has not yet developed. Either that, or the new drug has what appears to be superior pharmaceutical properties like fewer doses needed, oral versus intravenous, or fewer side effects. The challenge to antimicrobial research today is finding one to which bacteria are still susceptible. Resistance to antibiotics has reached a critical point.

        Not all bacteria behave the same way. Some are prone to acquiring and passing on antibiotic-resistance genes, others hardly. A good example are two organisms of pyogenic Gram-positive cocci, Staphylococcus aureus and Streptococcus pyogenes. Both can cause severe infections in humans. It wasn’t long after the introduction of penicillin that strains of S. aureus began to emerge that were resistant to penicillin at a very high level. They were found to produce an enzyme called penicillinase (now more appropriately called beta-lactamase) that destroyed the central part of the penicillin molecule, rendering it useless. This resistance mechanism spread like wildfire so that by the 1960s, just around half of all S. aureus isolates were resistant to penicillin and the related compound ampicillin. Today, over 90% of S. aureus is penicillin resistant.

            Contrast this with the other serious Gram-positive coccus, Streptococcus pyogenes. It has never developed resistance to penicillin. The drug works against it as well today as it did in 1945. That’s not to say resistance of this organism can never happen; S. pyogenes has developed resistance to other drugs. Antibiotic resistance is a very complex field, and clinical laboratories must be vigilant in keeping track of the problem so that some appropriate measures can be taken to contain it when it does occur.

          Hospitals keep track of the level of antibiotic resistance in their institutions by a tabulation known as an antibiogram. It is simply the number of antibiotic susceptible strains isolated over a defined period, usually a year, computed as a percentage of that organism's total number of isolates. For instance, if a hospital had 1,000 strains of Escherichia coli isolated from individual patients during the year from various sources such as urine, blood, and others, and 850 of them were susceptible to ciprofloxacin, the figure posted for that drug would be 85, for 85% of strains susceptible. These numbers are recorded and tracked through the years, and the results are worrying. The number of resistant strains doesn’t usually show a marked increase, but an increase nonetheless. The number susceptible may go from 85% one year to 83% the next, then 80%. And on and on. It’s like that for many antibiotics. Just where it all ends up is unknown, but the trend is unmistakable. The clock is ticking

            Some researchers have hinted that humans may be beginning to enter the “post-antibiotic era,” as the antibiotics available to us are no longer effective against a wide range of microbes.  We’re not there yet; it’s still very rare that a bacterium causing an infection is resistant to all available antibiotics, a condition called pan-resistance. Most bacteria are susceptible to a few antibiotics. The main problem with antibiotic resistance is that most infections are initially treated empirically; the physician uses the best “guesstimate” of the most likely successful drug-bug combination. Lab studies take at least a day, more likely up to two days, to come back, and if the guesstimate is wrong and the patient has a resistant microbe, that means the patient has gone a couple of days without appropriate therapy. In less severe infections, that may not matter too much. But it is life and death in acute infections involving sepsis and other serious symptoms—the more resistant the microbe, the more likely treatment failure in those initial “guesstimate” situations. Hospitals are encountering this phenomenon more and more each year.

          An unfortunate term that has come into vogue in recent years is “superbug.” The name conjures an image of this plague-like beast of a microbe that infects and destroys with impunity. That’s not it at all. The term superbug refers to microbes that have acquired resistance to a set of antimicrobial agents and therefore are difficult to treat in the initial stages of infection. They generally don’t possess any characteristics of virulence and infectivity that distinguish them from any other opportunistic pathogen. If a species of Klebsiella, a common Gram-negative rod, develops resistance to imipenem, ceftriaxone, levofloxacin, gentamicin, and piperacillin, all usually effective broad-spectrum antibiotics, that’s bad. Really bad. But a few antibiotics may still work, perhaps amikacin and/or colistin.  The issue is that those antibiotics that are effective are not first-line drugs and are administered after appropriate laboratory testing results are available. The organism has a leg-up, a couple of days to make its mischief before a proper drug is given. If the patient is badly compromised, that may be too late.  Sepsis can progress rapidly, and time is precious.

 

          The term “antibiotic stewardship” is a good one, encompassing the judicious use of antibiotics. This includes, of course, their use in hospitals and healthcare settings. But it also includes agriculture and industry. Several bacterial genes coding for antibiotic resistance in human pathogens have been found to have originated in organisms that do not cause disease and are not intimately associated with humans. But their genes were passed along to human pathogens somewhere along the way, and the chain continued. Clearly, it’s important that new chains of antimicrobial resistance are not allowed to happen, or at least minimized. This means that antibiotics only be used when absolutely necessary, with the correct drug selected for the job, and not used in excess.

            That’s easier said than done. Ask any primary care physician, and they will likely tell you that one of their biggest headaches is trying to convince their patient that an antibiotic will not cure their infection or condition. Sometimes an antibiotic is prescribed to make the patient happy. Some people don’t take all their antibiotics when prescribed, put the excess away in the medicine cabinet, then haul it out and “self-prescribe” when they feel sick. In some countries, antibiotics are available without a prescription; in others, they are sold on the black market. All of this adds to the problem of antibiotic resistance.        

            With antibiotic resistance becoming a larger and larger problem, some researchers have reverted to the once-popular philosophy of using the body’s defense mechanisms to thwart infections. So far, not much clinical success has met the effort. But research today is light years ahead of where it was just 50 years ago. Perhaps some viable alternative to antibiotics may one day be developed. We’ll just have to wait and see.

Multi-drug resistant Pseudomonas aeruginosa (PHIL)

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