The first steps to defining microbial virulence*

* This section was co-authored by Professor Gordon Dougan and Dr Lara Marks.

Right from the very earliest days of microbiology scientists knew that some bacteria were more likely to cause disease than others. Some bacterial species were so highly pathogenic that microbiologists referred to them as 'pathogens'. It soon became apparent, however, once microbiology was applied more broadly to studying healthy humans, that many bacteria rarely or never caused disease. Many harmless bacteria, for example, were found to live in the intestine and on the skin. Such bacteria were called 'commensals', reflecting the fact that they form part of a community that live on their host in a more or less balanced non-harmful relationship. Eventually scientists realised that even commensals often adapt to living on their host rather than free in the environment. This begged the question as to why some bacteria (and viruses and fungi) were 'virulent' and able to cause disease and why others did not (avirulent).

Early clues about virulence came from the observation that some microbes, such as Corynebacterium diphtheriae (the cause of diphtheria), produced potent toxins (poisons) that could cause illness or even disease symptoms. Bacteria released such toxins into the host's tissues where they could cause local or even systemic damage. Yet, not all 'pathogenic' or virulent microbes produced readily identifiable toxins, so virulence was clearly more complicated than this.

Figure 5.1: Photomicrograph of Gram-positive Corynebacterium diphtheriae. Credit: Centers for Disease Control and Prevention. First discovered in 1884, Corynebacterium diphtheriae are rod-shaped bacteria. Spread by person to person contact through respiratory droplets, the bacteria release a toxin which once absorbed can cause damage to mucous membranes in the tonsils, pharynx, larynx, and nose. This can lead to breathing difficulties and even death. If the toxin reaches other organs it can cause mycarditis (inflammation of the heart), paralysis and nephritis (inflammation of the liver). Diphtheria is one of the leading causes of childhood mortality around the world, especially in tropical regions and low-income countries. Up to ten per cent of those who contract diphtheria risk dying. Antibiotics and serum therapy are often useless for helping such cases. The disease can be prevented by vaccination (European Centre for Disease Prevention and Control).

Microbiologists also knew that it was possible to attenuate (weaken) the virulence of some pathogens by repeatedly culturing them in the laboratory. It was this approach that was used to create some of the first live vaccines. This included the tuberculosis vaccine (BCG), developed by the French bacteriologists Albert Calmette and Camille Guérin in the early twentieth century. They produced the vaccine by cultivating ever more weakened strains of the Mycobacterium bovis, the causative agent of tuberculosis, every three weeks. In all, they subcultured the bacillus 230 times to make the vaccine (Calmette).

Knowledge about virulence mechanisms was greatly aided by the emergence of the new field of microbial genetics after the Second World War. Research in this area provided the first evidence that microbes actually harbour genes that encode their genetic characteristics. It also provided invaluable genetic tools for unpicking the process of virulence. Among the most helpful methods for dissecting virulence was to recombine genes between microbes, which took advantage of the process of conjugation, high-frequency recombination transfer and transduction.

By the 1950s the genetic mechanisms by which bacteria become resistant to antibiotics had become a hot research topic, prompted in part because of concerns about the rapid rise of bacterial resistance to new antibiotics. Each time a new antibiotic was released as a medicine, antibiotic resistant bacteria seemed to be ready to respond, often within a year or two of the commercial release of the antibiotic (see figure 3.6).

Steady work throughout the 1950s and the early 1960s linked many antibiotic resistance genetic determinants to small extrachromosomal DNA elements, known as plasmids, picked up by the bacteria. One such plasmid was the F 'fertility' factor, first identified by Esther Lederberg in 1952. Thereafter, many more different types of plasmids began to be discovered in a variety of different bacterial species, many of which were pathogens. Such plasmids lay at the heart of the growth of microbial genetics as a new discipline and the rapid rise of expertise in handling them.

Click here for more information on Lederberg’s discovery of the F factor.

One of the key tools the microbiologists had at their disposal for investigating virulence was serotyping. Adopted on a wide scale from the late 1920s, this method uses antibodies to detect variable structures (antigens) found on the surface of bacteria. Some early attempts were made to link virulence to particular serotypes, but this approach was not clear cut as can be seen from the case of Escherichia coli. Originally the species of choice for geneticists, because it was a common commensal of the intestinal tract, serotyping initially indicated that E. coli did not appear to be particularly associated with virulence, as was the case for Salmonella and Shigella. Over time, however, it became clear that some E. coli strains were a likely suspect in a number of disease syndromes, such as scouring or diarrhoeal disease in neonatal pigs and cattle as well as so called 'traveller's diarrhoea' suffered by tourists who went abroad.

Click here for more on the history of serotyping

Figure 5.2: Coloured electron micrograph of Enterotoxigenic E coli. Credit: NIAID. These bacteria are a major cause of diarrhoeal disease in lower-income countries, especially among children, and a leading cause of traveler's diarrhoea.

Work in the early 1960s started to implicate certain heat-labile antigens - antigens that could be destroyed or altered by heat - as possible factors facilitating the attachment of E. coli to farm animals in a host restricted manner. In 1966 one such antigen, known as K88, associated with pig E. coli, was shown by Ida and Fritz Orskov to be encoded on a plasmid (Stirm, Orskov, Orskov). Two years later. a team of international researchers studying samples taken from patients who had been struck by cholera during an an outbreak in Kolkata, India and Dhaka, Bangladesh (then East Pakistan, identified two types of toxin, known as Heat-stable (ST) or heat-labile (LT), that were produced by similar E. coli, which were consequently named enterotoxigenic E. coli (ETEC) (Sack, 1968; Sack 2011). The genes for both ST and LT were found to be carried on plasmids. Many ETEC bacteria produced ST alone, while others produced both ST and LT from F-like plasmids. Later studies found the two toxin genes on a variety of different plasmid types. Additionally, other adhesion or colonisation factors such as K99 (pigs/cattle) and CFA1 (humans) were found and also shown to be plasmid encoded (Elwell). These fortuitous observations helped lay the foundation for the first bacterial genetic studies around virulence.

Figure 5.3: Ida and Fritz Orskov, 28 July 1957, Credit: Esther Lederberg Memorial Website. Ida (née Oppenheuser) (1922-2007) was a Danish physician and bacteriologist. She first attracted scientific attention through her thesis on Klebsiella, a Gram-negative, rod-shaped bacteria. Published in 1956, Ida's work was one of the first scientific studies to demonstrate the presence of bacterial cross infections in hospitals. Ida acquired her interest in bacteriology from Fritz (1922-2015), a fellow student studying medicine and for a doctorate at the University of Copenhagen. The two of them married in 1948 and together ran a research centre focused on diseases caused by Enterobacteria at the Danish Serum Institute (State Serum Institute) where Fritz’s father, Jeppe, was once the director.

One of the key pioneers behind the development of genetic studies for virulence was Herbert Williams Smith. A modest British Veterinarian turned researcher, Smith combined his veterinary pathological knowledge with well-designed experiments to understand the genetic basis of bacterial infections. Among his greatest contributions was to exploit plasmid encoded determinants to define the genetic basis of ETEC pathogenesis. His experiments provided a model for much future work in the field.

Smith is renowned for the seminal experiments he conducted with his colleague Margaret Linggood in the early 1970s. Together they constructed a series of E. coli bacteria that incorporated DNA from other bacteria via conjugation (transconjugants) that expressed either K88, LT or both K88 and LT. They also made these transconjugate combinations in different commensal E. coli recipient strains. Upon feeding cultures of these transconjugates to neonatal pigs they found that those given K88 producing E. coli developed mild diarrhoea whereas those that received the E. coli producing both K88 and LT developed severe diarrhoea and full disease. Pigs receiving E. coli producing LT but not K88 did not develop diarrhoea and poorly colonised the small intestine of pigs. From this it appeared that K88 and LT producing E. coli K12 did not cause disease. Critically, Smith and Linggood demonstrated that expression of a toxin alone was not sufficient to convert a commensal E. coli into a pathogen. An adhesin factor was also required. Also, other E. coli factors, in addition to K88 and LT were required for the expression of full disease. For example, they showed that the laboratory strain E. coli K12 was a poor coloniser of the intestine (Smith, Lingwood).

Figure 5.4: Herbert Williams Smith (1919-1987), credit Royal Society. Smith was born in Wales and undertook his PhD research training at the London School of Hygiene and Tropical Medicine before joining the then Animal Health Trust where he ran a small research team for three decades that never numbered more than one or two people. He subsequently spent the rest of his career investigating the genetics of bacterial infections and antibiotic resistance in farm animals. Smith was the star of a generation of talented experimental scientists who worked with limited resources but on real economic and social problems through the application of basic science principles. Drawing on his experience as a veterinary scientist he combined excellent animal management with brilliant experimental design. His approach involved inspecting the corpses of many diseased animals brought in from surrounding farms and experiments with small groups of real farm animals to answer challenging questions. Based on this work he made many significant breakthroughs about how E. coli causes disease. For example, he was responsible for the discovery of the ST toxin of ETEC and showing this toxin, E. coli haemolysin, and K99 was carried on plasmids. He also characterised the E. coli verotoxin. In addition, he helped to improve knowledge about Salmonella and Staphylococcus diseases and the mechanism behind antibiotic resistance. Based on his work on AMR, Smith was one of the first scientists to urge governments to limit the use of antibiotics in farm animals. On a practical level he also demonstrated the value of phage therapy in controlling animal diseases.

Figure 5.5: Table V from Smith, Lingwood seminal paper, in 1971, which reported a series of remarkable experiments pigs that was arguably the first real use of genetics to prove the genetic basis of virulence in a pathogen, Enterotoxigenic E. coli. The tables dramatically show that the acquisition of an adhesin (K88) and a toxin (LT) by a commensal E. coli can convert it into a 'pathogen'. Also, expression of the potent toxin itself is not sufficient to endow full virulence on the commensal.

Interestingly, Smith and Lingwood reported their seminal experiments in 1971, just as the era of recombinant DNA was dawning. This technology, applied by others, soon transformed knowledge about bacterial virulence at the genetic level. Among those who adopted the new technique with alacrity were the American bacterial geneticists Louis S Baron, Stanley Falkow (1934-2018) and Sam Formal at the Walter Reed Army Institute of Research (WRAIR) in Bethesda, Maryland. The three researchers focused their efforts on bacteria that live in the intestine that cause disease, known as enteric pathogens, including Salmonella Typhi (typhoid), Shigella flexneri (dysentery) and E. coli. Believing that pathogenicity might be linked to some sort of genetic adaptation for survival, the three researchers set out to find out the extent to which pathogenic enteric bacteria were genetically different from their non-pathogenic relatives. Their hypothesis went against conventional thinking at the time, which envisaged pathogens as degenerate forms of bacteria that grew at the expense of the host and caused damage in the process.

Figure 5.6: Photograph of Stanley Falkow, credit Stanford University. An American bacterial geneticist, Falkow (1934–2018) devoted his career to understand the process by which bacteria cause disease. He studied a wide variety bacteria, from diarrhoea-causing E. coli Salmonella to bacteria that cause whooping cough and bubonic plague. Using emerging microbial genetic technologies he demonstrated how genes from disease-causing bacteria could change benign bacteria into harmful ones and advanced the understanding of of the molecular structure of plasmids and jumping genes involved in the transmission of AMR. Based on his findings, he pressed the US Food and Drugs Administration to remove antibiotics from animal feed. He also showed that a sub-type of E. coli was responsible for the life-threatening diarrohea prevalent in many developing countries (Anon).

To get the ball rolling the WRAIR researchers created a number of hybrid bacteria with the help of bacteria known as 'Hfr crosses'. First developed by two French geneticists, François Jacob and Elie Wollman in the late 1950s, such bacteria are created with the help of F-plasmids. Carrying a fertility (F) factor, these plasmids can induce mating or conjugation between F-positive donor bacteria and compatible F-negative recipients. The F factor prompts the formation of a mating pair by constructing a pili structure at the donor cell surface. Once formed, the F factor will initiate self-replication and transfers a copy of itself into the recipient cell. Occasionally, the F factor plasmid can integrate into the donor cell chromosome and if mating is initiated, then sections of the donor cell chromosome are mobilised or transferred during conjugation into the recipient cell where they will recombine with the recipient chromosome if sufficiently related (Figure 5.7).

With the help of the Hfr crosses, Falkow and Formal created a number of E. coli and Shigella hybrids that were attenuated for virulence and shared genetic characteristics of both species. The two scientists used E. coli Hfr strains as donors and either Shigella flexneri or Salmonella Typhi as recipients. They hoped that the resulting hybrids would not only provide clues to understand the genetic factors that underlay virulence, but also prove useful to create live oral vaccines for typhoid and dysentery. Falkow and Formal were able to identify recombinant Shigella that harboured E. coli metabolic markers and used these to vaccinate animals such as guinea pigs and monkeys (Formal, LaBrec Palmer, Falkow). Although their approach failed to deliver a vaccine it laid an important foundation for future genetics studies on virulence.

Figure 5.7: Diagram of Hfr conjugation. Credit: AC Shrader, Wikipedia. Key to the number is set out below.

Key to the numbers in the Hfr diagram above.

  1. The insertion sequences (yellow) on both the F factor plasmid and the chromosome have similar sequences, allowing the F factor to insert itself into the genome of the cell. This is called homologous recombination and creates an Hfr (high frequency of recombination) cell.
  2. The Hfr cell forms a pilus and attaches to a recipient F- cell.
  3. A nick in one strand of the Hfr cell’s chromosome is created.
  4. DNA begins to be transferred from the Hfr cell to the recipient cell while the second strand of its chromosome is being replicated.
  5. The pilus detaches from the recipient cell and retracts. The Hfr cell ideally wants to transfer its entire genome to the recipient cell. However, due to its large size and inability to keep in contact with the recipient cell, it is not able to do so.
  6. a. The F- cell remains F- because the entire F factor sequence was not received. Since no homologous recombination occurred, the DNA that was transferred is degraded by enzymes.
  7. b. In very rare cases (6.b.), the F factor will be completely transferred and the F- cell will become an Hfr cell.

    8. References

    Anon (9 May 2018) 'In Memoriam, Stanley Falkow', IDSA News.Back

    Calmette, A (1931) 'Preventive vaccination against tuberculosis with BCG', Proceedings of the Royal Society of Medicine, 24, 85-94.Back

    Elwell, LP (1980) 'Plasmid-mediated factors associated with virulence of bacteria to animals', Annual Review Microbiology, 34, 365-96.Back

    European Centre for Disease Prevention and Control, 'Fact sheet about diphtheria'.Back

    WH (1979), ‘Contributor to systematic microbiology’, Fritz Kaufmann, International Journal of Systematic Bacteriology, 29/4, 47.Back

    Formal, SB, LaBrec, EH, Palmer, A, Falkow, S (1965), 'Protection of monkeys against experimental Shigellosis with attenuated vaccines', Journal of Bacteriology, 90/1, 63-68.Back

    Sack, RB (1968), 'Diarrhea producing factors in cultures of Escherichia coli', Proceedings of the 4th Joint Conference, Japan-U.S.Cooperative Medical Science Program, Unzen, Japan, 23-5.Back

    Sack, RB (2011), 'The discovery of cholera - like enterotoxins produced by Escherichia coli causing secretory diarrhoea in humans', The Indian Journal of Medical Research, 134/2, 171-78.Back

    Smith, HW, Lingwood, M (1971) 'Observations on the pathogenic properties of the K88, Hly and Ent plasmids of Escherichia coli with particular reference to porcine diarrhoea', Journal Medical Microbiology, 4, 467-85.Back

    Stirm, S, Orskov, I, Oskov, O (1966) 'K88, an episome-determined protein antigen of Escherichia coli', Nature, 209/5022, 507-8.Back

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