Just over 2000 bacterial species have been cultured at least once from clinical specimens, and more species undoubtfully remain to be cultured. The same enumeration remains to be performed for viruses and microeukaryotes in human microbiota . Culturing a microbe remains the goal in microbiology, as the cultured microbe is an unsurpassed starting point for obtaining knowledge, opening the door to fully understanding and eventually manipulating the microbe, including prevention of colonization and disease through vaccination and treatment. Accordingly, pathogens were among the very first microbes to be cultured, and in this thematic issue of Clinical Microbiology and Infection we tell the story of the discovery of four such big killers, illustrating the sometimes tortuous pathway of microbial discovery. Thèves et al.  report on the discovery of smallpox, and Cambau , Lippi  and Butler  report on the discovery of Mycobacterium tuberculosis, Vibrio cholerae, and Yersinia pestis, respectively.
The four stories provide lessons on which to base our research in clinical microbiology. In no case did the initial discovery of the pathogen arrive from nowhere; it depended on a previous corpus of popular, empirical and scientific knowledge. In particular, these four quite different infectious diseases had been recognized for centuries as specific entities by populations and doctors, through a combination of epidemiological and clinical observations, such as epidemics and swollen lymph nodes (buboes) in the case of plague . Paleomicrobiology, which is concerned with the retrospective history of microbes and the diseases that they caused , now allows us to appreciate this aspect, as smallpox virus , M. tuberculosis  and Y. pestis  have been detected in suitable ancient specimens, being, respectively, the skin, bones, and dental pulp. V. cholerae has been detected in ancient specimens, using the suitable material (the intestinal contents of cholera victims) .
Discovering a new pathogen has often relied on the development and use of new technology. Microscopy, more precisely microscopy improvement, contributed to the discovery of M. tuberculosis by Robert Koch in 1882, as he invented a new staining method ; and the use of electron microscopy was decisive in the case of smallpox . Interestingly, in 1854, both John Snow in London and Filippo Paccini in Florence also used optical microscopy as part as their investigation of cholera epidemics, but failed to go further in culturing V. cholerae, which was finally isolated by Koch 30 years later . Indeed, the use of solid culture medium instead of broth, previously developed in Louis Pasteur's laboratory, was a decisive step in the initial isolation of M. tuberculosis and V. cholerae by Koch [3, 4]. For plague, the use of an ambient temperature instead of 37°C contributed to the success of Alexandre Yersin in 1894, in addition to the culture of diseased lymph nodes instead of blood . However, two major pathogens, i.e. Treponema pallidum and Mycobacterium leprae, remain to be cultured in axenic medium, as they have been propagated only in animals. Today, the discovery of microbes has moved towards organisms in complex microbiota, including bypassing organisms, resident organisms, and opportunistic pathogens. Bacterial culturomics, using diversification of culture media and culture conditions (atmosphere and temperature of incubation), now yields hundreds of colonies per culture plate to be identified . This rebirth of culture has been made possible by a new technology for the rapid identification of such huge numbers of colonies, matrix-assisted laser desorption ionization time-of-flight mass spectrometry . Therefore, more and more organisms are being discovered together, cultured in the same laboratory from the very same specimen, in contrast to the singular history of their predecessors.
Also, the four histories reported in this thematic issue teach us to avoid some of the pitfalls of scientific communication in microbiology, the last but not least step of microbe discovery. The dispute over who was the very first discoverer of Y. pestis, after the controversial 1894 paper by Shibasaburo Kitasato in the Lancet, is well known [5, 13]. A rush to publish before a pure culture of the new microbe had been obtained, and mixing his own data with those of another doctor, led Kitasato to publish inaccurate data, and finally tarnished the glory of the discoverer of Clostridium tetani. The influence of Kitasato's reputation, preventing a neutral review of the data by the Lancet, also had a negative role in this history . Ignoring and under-reporting previously published data is another pitfall when dealing with microbe discovery. Even the Nobel prize winner Koch failed to mention previous work by Jean Antoine Villemin , who, in 1865, established the transmissibility of tuberculosis, in his very first paper on M. tuberculosis ; Koch finally acknowledged this contribution in his second paper, published 2 years later in 1884 . Likewise, Koch clearly missed previous work by Paccini regarding the discovery of V. cholerae .
In conclusion, the four microbe discovery stories reported here are a source of lessons for microbiologists searching for new microbes. The use of new laboratory tools and new concepts is a source of discovery. Fair reporting of unique discoveries, using modern standards of species definition and modern tools to communicate, is a source of durability.