Unravelling the biological mechanism behind AMR

How microbes develop resistance to drugs was a question that puzzled many researchers from the early twentieth century. For much of the 1920s and 1930s the ability to address the issue was confounded by a belief that bacteria were simple organisms that multiplied by dividing. On top of this, scientists had no idea that bacteria could possess genetic material and little understanding of the hereditary role DNA played. Bacteriologists generally assumed that bacteria acquired new traits through a process called 'training' or 'adaptation'. This belief was reinforced by experiments conducted by scientists in Cambridge in the 1930s. Such work showed bacteria to be capable of adapting to different nutritional environments by synthesising completely new enzymes, and that they could pass on this characteristic to subsequent generations. Scientists attributed the phenomenon to dietary changes, which was conceived of within a purely physico-chemical framework that bore no relation to genetics (Creager).

Figure 4.1: Cyril N Hinshelwood, 1931 (Unknown photographer, credit: Wikipedia). Hinshelwood (1897-1967) was born in London and completed a degree in physical chemistry at Oxford University. After working as a chemist in an explosives factory during World War I, Hinshelwood became a tutor and then a professor in chemistry at Oxford University. He was later the president of the Royal Society. Renowned for his skill in planning experiments, Hinshelwood used simple equipment to answer complex questions. A great deal of his research was dedicated towards determining the relationship between the speed of a chemical reaction and factors such as concentration, temperature or the presence of foreign substances. Hinshelwood's early work, for which he shared the Nobel Prize in 1956, concentrated of the explosive reaction of hydrogen and oxygen. He later chose to study bacteria because they could bring about chemical reactions of 'amazing variety and subtlety in extremely short time, and they manifest the characteristics of living things in one of the simplest possible forms, thereby making some of the mysteries of life more easily accessible to experiment' (Hinshelwood).

The theory of adaptation helped to explain bacterial resistance to antibiotics in many studies in the 1940s. A key figure in this research was Cyril Hinshelwood, a British scientist who built up a reputation for elucidating chemical chain reactions in the 1920s. From 1940 onwards Hinshelwood undertook a series of experiments to investigate the responsiveness of bacteria to their environmental conditions. His research concentrated on the bacterium lactis aerogenes (Aerobacter aerogenes), a non-pathogenic organism sometimes involved in the souring of milk. Hinshelwood managed to induce a stepwise progression of antibiotic resistance in the bacteria by exposing them to increasingly higher concentrations of different antibacterial agents, including proflavine sulphate, propamidine, chloramphenicol and 8.Hydroxyquinoline.

Overall Hinshelwood concluded that bacterial resistance to drugs originated from two distinct sources. The first was rooted in the selection of abnormal individuals or mutants. The second was caused by the drug inducing more or less permanent changes to the chemical reactions within bacterial cells (Dean, Hinshelwood; Creager; Thompson).

By the late 1940s a number of discoveries had been made about bacteria and the inheritance process which undermined the adaptation model. The first challenge came from the work of Max Delbrück and Salvador Luria. In 1943 they began a series of experiments based on some earlier observations made by Felix d'Herelle, a French-Canadian microbiologist. He had noted in the 1920s that a culture of bacteria became clear whenever it was exposed to a bacteriophage (phage), a type of virus that infects bacteria, and then returned to cloudy some time later. Scientists assumed that the trend observed by d'Herelle was down to the bacteria acquiring resistance to the phage and repopulating the culture. Both Luria and Delbrück wanted to find out whether such resistance stemmed from acquired immunity, induced by the viral infection, or instead came from a spontaneous mutation that existed prior to exposure to the phage.

Figure 4.2: Max Delbrück (above) and Salvador Luria (below) at Cold Spring Harbor Laboratory, 1941 (Credit: National Library of Medicine). Delbruck (1906-81) was born in Berlin, Germany. He originally trained in astrophysics and then switched to biological research in the 1930s. In 1937 he decided to leave Germany because of the Nazi regime. He took up a Rockefeller fellowship at the California Institute of Technology where he began researching bacteria and their viruses (phages). Luria (1912-91) was born in Turin, Italy, where he trained in medicine. Banned from taking up an academic fellowship because he was Jewish, Luria left his home country in 1938 and in 1940 received a visa to take up a Rockefeller foundation fellowship at Columbia University in New York. The two scientists first met on New Year's Eve in 1940 and spent the following summer together at Cold Spring Harbor Laboratories during which time they began their first experiments.

Figure 4.3: The hypotheses tried out in the Luria-Delbrück distribution experiment (Credit: Bois).

Luria and Delbrück's experiment (figure 4.1) involved growing up a small number of parallel cultures of Escherichia coli bacteria in liquid in small vials and then placing equal volumes of these separate cultures onto agar plates seeded with a type of phage called T1. A few days later the two scientists counted the number of colonies of resistant bacteria found on the plates. They predicted that if the acquired immunity hypothesis was correct, low numbers of resistant colonies would appear on the plate. This was because it would take time for the bacteria to acquire resistance to the phage. By contrast, if the mutation theory prevailed, there would be wildly varying numbers of resistant colonies. Much to Luria and Delbrück's surprise, however, they found that the number of resistant colonies varied dramatically on each plate. This suggested that random mutation, rather than acquired immunity, was in operation (Bois; Murray).

One of the first to seize upon Luria and Delbrück's finding was Milislav Demerec, director of the Department of Genetics at Cold Spring Harbor. In 1944 he modified Luria and Delbrück's experimental method to study the origin of resistance of Staphylococcus and E. coli bacteria to penicillin. His experiments suggested resistance was 'not induced by the action of penicillin on bacteria' but arose 'independently by mutations'.

Overall Demerec believed that penicillin acted as 'a selective agent to eliminate non-resistant individuals'. Based on his findings Demerec concluded resistance to be 'a complex characteristic, and that it must involve a number of mutations; if it is assumed that genes are responsible for these mutations, a number of genetic changes must be involved' (Demerec).

Figure 4.4: Milislav Demerec, n.d. (Credit: Cold Spring Harbor Laboratory Archives, New York). Demerec (1895-1966) was born in Kostajnica, a small town in central Croatia. He completed his undergraduate studies in agriculture in his home country and then did a doctorate at Cornell University, in New York, before joining the staff of the Department of Genetics at Carnegie Institution of Washington in Cold Spring Harbor in 1923, rising to become its director two decades later.

Demerec devoted his career to getting a better understanding of genes, their structure and function and their spontaneous and induced mutability. Initially his research looked at the genetics of maize and then he switched to looking at Drosophila, the common fruit fly. He turned to working on the genetics of bacteria and their viruses after learning about about Luria and Delbrück's work. His research involved investigating the induction of genetic mutations by ultraviolet radiation and neutrons. During World War II he deployed his knowledge about mutagenesis to induce a mutant strain of Penicillium (X-1612) to increase the yield of penicillin.

As a result of his research into AMR, Demerec established two key rules for administering antibiotics. Firstly, initial doses of the drug needed to be high enough to prevent the occurrence of resistant bacterial mutants. Secondly, it was important to use a combination of antibiotics and not just one singly, because a mutant strain resistant to one antibiotic had little chance of simultaneously being resistant to another one (Glass).

The comments made by Demerec in 1945 are particularly striking because until then scientists had assumed bacteria did not have any genes. New evidence, however, begun to surface to undermine this viewpoint. The first proof that bacteria contained genetic material came to light through the research of Oswald Avery and his coworkers Colin MacLeod and Maclyn McCarty at the Rockefeller Institute for Medical Research. In late 1943 the team launched some experiments to determine the mechanism by which previously harmless pneumococcal bacteria could turn into more virulent forms capable of causing disease. This phenomenon had first been noted back in 1928 by the English bacteriologist Frederick Griffith. From their experiments, Avery and his collaborators made the surprising discovery that the transformation of the bacteria appeared to be triggered by deoxyribonucleic acid or DNA (Avery, MacLeod, McCarty). Their finding challenged the conventional wisdom of the time that DNA was a relatively unimportant chemical substance and the assumption that proteins were the transmitter of genetic traits. Click here for more information on the experiments conducted by Griffiths and Avery.

Published in February 1944, the Avery team's results immediately grabbed the scientific community's attention. This was because it was the first time any scientist had demonstrated that DNA could bring about an enduring, heritable change in an organism. Some of the excitement that their finding generated was expressed by William Astbury, an English physicist and molecular biologist. As he put it to a friend in October 1944, it was 'one of the most remarkable discoveries of our time… I wish I had a thousand hands and labs with which to get down to the problems of proteins and nucleic acids. Jointly those hold the physico-chemical secret of life, and quite apart from the war, we are living in a heroic age, if only people can see it' (Cobb).

Click here for more about William Astbury.

Another one of those electrified by the results from Avery's team was Joshua Lederberg, a 19 year old then studying for a doctorate of medicine (MD) at Columbia University and working with Francis Ryan who was investigating the problems of bacterial mutation and replication at the molecular level. For Lederberg the new findings opened up unlimited opportunities to probe the chemical nature of the gene. In April 1946 he initiated a series of experiments to see what he could learn about genetic exchange in bacteria. At this point the conventional wisdom was that bacteria were simple organisms that replicated by dividing into two genetically identical daughter cells. Wanting to test this assumption, Lederberg designed some experiments to determine whether bacteria displayed any sexual behaviour and recombined their genes in the reproduction process (Bodmer).

Figure 4.5: Joshua Ledeberg's excited reaction to the Avery paper as recorded in his diary. (Credit: National Library of Medicine). The entry reads '20 Jan 1945, 2100 Saturday. I had the evening all to myself, and particularly the excruciating pleasure of reading Avery '43 on the deoxyribose nucleic acid responsible for type transformation in Pneumococcus. Terrific and unlimited in its implications. Viruses are gene-type compounds, but they cannot grow on synthetic or even dead media, and their capacity for production is limited to reproduction. The TF of Pneumococcus has every characteristic of a mutation. The obvious questions still to be considered are the fraction of serum that is involved in the reaction system; the induction of mutation in the TF by use of x-ray and more controllable methods; the problems of its antigenic specificity and relations to the specific polysaccharide whose manufacture it regulates or initiates. Also the possibility of activity of TF in vitro or in killed systems must be investigated, although the presence of phosphatases and deoxyribonucleases present a difficult problem. I can see real cause for excitement in this stuff though.'

Figure 4.6: Joshua Lederberg in a laboratory at the University of Wisconsin, Oct 1958 (Credit: University of Wisconsin). Lederberg (1925-2008) was born in Montclair, New Jersey. Following his work with Tatum, Lederberg completed a PhD at Yale University and took up a position at the University of Wisconsin-Madison in 1947 where he launched research into bacterial conjugation in Salmonella.

Figure 4.7: Edward L Tatum, photographer and date unknown (Credit: Wikipedia). Born in Boulder, Tatum (1909-75) earned a doctorate in biochemistry from the University of Wisconsin in 1934 before taking up a position at Stanford University. Together with George Beadle, he demonstrated, through experiments with Neurospora crassa, a type of pink bread mould, that genes control all the biochemical processes in organisms. In 1945 he moved to Yale University and it was here that he and Joshua Lederberg did their work together.

To conduct his experiments, Lederberg sought help from Edward Tatum, an expert in bacteriology at Yale University. Tatum had importantly developed two different bacterial mutants derived from the K-12 strain of E. coli that were ideal for testing the ability of bacteria to mate. Each strain was unable to synthesise certain nutrients necessary for growth. After mixing the two strains together and placing them in incubation, the two scientists were surprised to see a number of colonies growing. They were startled because they had not added any nutrients to help the bacteria grow. Moreover, all the individual bacteria within the recombinant colony had the same genetic profile. Some also appeared to be resistant to phage T1, another trait inherited from one of the parental strains. Based on these results Lederberg and Tatum concluded that the bacteria had exchanged genes and passed on certain traits to successive generations, a process called 'conjugation' (Tatum, Lederberg).

Figure 4.8: Lederberg and Tatum's experiment.

Having detected bacterial conjugation in E. coli, Lederberg decided to undertake further investigations to see if the same process could also be observed in another species of bacteria - Salmonella Typhimurium. This he did with a number of other colleagues. One of his key collaborators was Esther Lederberg (nee Zimmer), his wife, who was conducting her own pioneering research on bacterial genetics and new techniques to do this. Click here for more information about Esther Lederberg's work.

Figure 4.9: Electron micrograph of Salmonella Typhimurium (stained red) invading cultured human cells (Credit: Rocky Mountain Laboratories, NIAID, NIH). Salmonella is a cousin of E. coli. It typically lives in animal and human intestines and is responsible for 1 in 4 global causes of diarrhoeal disease. Humans commonly get infected with the bacteria through contaminated water or food. Most cases of Salmonella infection are mild, but sometimes the symptoms can be life-threatening.

Figure 4.10: Joshua and Esther Lederberg in their laboratory in Madison, Wisconsin, 1958 (Credit: University of Wisconsin). The couple met shortly before Esther completed her master's degree in genetics at Stanford University and Joshua was doing his doctorate at Yale University. They married in 1946, just five months after their first meeting, and then moved together to the University of Wisconsin where Joshua had just been appointed a professor. Once in Wisconsin Esther worked as an unpaid research associate in Joshua's laboratory while getting her doctorate.

From 1947 onwards Lederberg's team undertook a series of experiments with several mutants of Salmonella. The team developed a number of tools to assist with the work. This included the cultivation of bacteria with two key genetic markers. The first marker was the inability to synthesise certain enzymes. The second was resistance to two antibiotics - penicillin and streptomycin. Such markers were chosen to make it easier to identify when the bacteria mated and exchanged genes. In addition, Esther had invented a new technique that made it possible to copy the exact pattern of microbial growth from one initial agar plate to others. Called replica plating, Esther's method (figure 4.3) enabled the rapid screening of a large number of individual isolated bacterial colonies based on their different observable characteristics, such as their nutritional requirements or sensitivity to antibiotics. Replica plating also made it easier to pick up and track any mutants (National Library of Medicine).

Figure 4.11: Diagram showing how replica plating works.

Figure 4.12: Photograph of Norton Zinder by Robert Reicher, 1992 (Credit: The Rockefeller University). Zinder (1928-2012) was born in New York City and joined Joshua Lederberg's laboratory's work on bacterial conjugation in 1942 after completing his undergraduate degree in biology at Columbia University. Zinder and Joshua Lederberg worked out the process of transduction after Zinder noticed that some of the mutant Salmonella bacteria he developed for experiments to investigate bacterial conjugation had not behaved as expected.

Based on the Lederberg group's efforts, it soon became clear that conjugation was not the only mode by which bacteria exchange genes. In 1951 Lederberg and one of his graduate students, Norton Zinder, discovered that genetic fragments could be transferred between bacterial cells via phages. They called the process 'transduction', based on the Latin verb 'transducere' meaning 'to lead across'. The discovery of transduction was of fundamental importance to understanding AMR because it helped to explain how bacteria acquired genes that made them resistant to drugs more quickly than happened in the case of genetic mutation and natural selection. Bacteria were simply able to pick up resistance genes from another strain via a phage. A year later the two Lederbergs conducted an experiment that demonstrated many bacteria could spontaneously pick up resistance to an antibiotic without prior exposure to the drug (National Library of Medicine).

By the mid-1950s three key molecular pathways had been worked out by various scientists for the transfer of genes between bacteria: transformation, conjugation and transduction. This process took place horizontally between bacteria. It was not the only way genes transferred. Mutations conferring resistance to antibiotics were also known to transfer vertically as a result of bacteria reproducing through binary fission. Figures 4.13 and 4.14 summarise the different ways genes were now understood to transfer horizontally and vertically between bacteria to confer resistance. The new understanding not only laid an important foundation for understanding how bacteria developed resistance to different drugs, but also provided important techniques for tracking antimicrobial resistance in the future.

Figure 4.13 - Vertical gene transfer between bacteria.

Figure 4.14 - Horizontal gene transfer between bacteria.

Figure 4.15 - Timeline of key discoveries in bacterial genetic exchange 1928-51.


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