Vladimir Sertić: forgotten pioneer of virology and bacteriophage therapy

Vladimir Sertić was a pioneer of bacteriophage research in the period between the two world wars. He was born and educated in Croatia, where he made his initial discoveries, and joined Félix d'Herelle's Laboratoire du Bactériophage in Paris in 1928. Original documents and a box with hundreds of sealed bacteriophages samples were kept in Sertić's Zagreb home for decades. Following Vladimir's death, his sister passed this archival material to Professor Zdravko Lacković in 1989. Some years later, these artefacts were opened and studied. Additionally, we conducted a literature search using the term ‘Vladimir Sertić’ in the databases PubMed and Google Scholar. After a detailed examination of these data, we established a chronology of his work and compiled a list of his scientific publications. A complete bibliography, with the exception of those publications already cited here, is provided as an appendix. Sertić's key contributions included the exploration of the properties of phage lysins, the devising of a uniform bacteriophage classification system and, in collaboration with his protégé, Nikolai Boulgakov, the isolation of numerous bacteriophage strains, including the famous φX174. Finally it was Sertić's pioneering work in Zagreb that offered confirmation that phages are live agents.


INTRODUCTION
Because of the growing problem of antibiotic resistance of bacteria, there has been a recent revival of interest in bacteriophage therapy. 1 This emerging interest in those bacterial viruses in the biomedical community has brought the topic to a wider professional and general public audience. Whether one takes Frederick William Twort's first inconclusive finding published in 1915 or the more precise and insightful publication in 1917 by Félix Hubert d'Herelle to be the exact 'birthdate', 2 phage research has a century-old history. 3 Although the research and lives of d'Herelle and the Georgian scientist George Eliava are described and known, 4 the two other important pioneers of bacteriophage research, Nikolai Boulgakov and especially Vladimir Sertic, have remained practically anonymous (figure 1).
Vladimir Sertićwas a Croatian microbiologist who in the later phase of his career held the position of scientific director at d'Herelle's private institute in Paris, the Laboratoire du Bactériophage, and whose scientific contribution to research and application of phage therapy and lysins was only partially described in Croatian-language publications. 5 Boulgakov (also spelled as Bulgakov or Buljgakov), the younger brother of the famous writer Mikhail, had left his homeland in Ukraine in 1922 because of the communist revolution, and settled in Zagreb. He took Yugoslavian citizenship and continued with his medical studies at the School of Medicine. 6 Upon graduation, Nikolai joined Sertićas an assistant, and eventually followed him to Paris. Together they co-authored 20 papers, including those mentioning the famous wX174 virus particle. Apparently, it was first isolated and named by Sertićand Boulgakov in their own comprehensive and original bacteriophage classification. 7 Moreover, Sertićhimself was one of the first investigators to focus heavily on the properties and nature of phage (bacterio)lysins: bacteriolytic enzymes produced by phages. His findings were the first to correctly show that lysins were actual enzymes, produced and encoded by biologically active phages. 8 These results were also in line with d'Herelle's claim that phages were, indeed, live agents. 9 This story of their work would not have been uncovered had there not been an effort made by one of the authors (Lackovic) from the Department of Pharmacology at the University of Zagreb School of Medicine. When preparing a manuscript in the late 1980s on scientific citation metrics from the affiliates of the School of Medicine before World War II, he encountered an unfamiliar name-that of Vladimir Sertic. 10 At that time, it was barely possible to find anyone even recognizing the name of the late Professor Sertic, let alone able to provide any details about his life and work. Eventually, Lackovićwas able to locate his sister, Dr Mira Sertic, who was willing to make her brother's papers and records available. Among these private documents were multiple handwritten manuscripts, and final printed publications. Most exciting was a box dated 1930, packed with 520 authentic commercial ampoules containing phages produced in d'Herelle's laboratory, with 157 additional agar-filled vials, each with a culture of a named specific strain of bacteria-probably a reference culture collection for research purposes (see figures 2 and 3). During his student days, he volunteered as a physician assistant at a military hospital in Zagreb, where his father was working. Assigned to bacteriology and venereal diseases wards, he was exposed to the evolving field of immunology and infectious diseases from his early student days. Thus, by the time of his graduation from medical school in 1925, he already had considerable laboratory experience.
After a prolific career as a researcher in Paris, Sertićreturned to Zagreb in 1939, where, because of the outbreak of World War II, he accepted a post at the university's School of Medicine. Unfortunately, in 1943, just two years into his appointment as a professor in the Department of Bacteriology, Serology and Immunology, he was struck with a serious combination of pneumonia, heart failure and rheumatism. Ultimately, he suffered an unidentified psychiatric episode and, although he recovered his mental health during the following year, he was still afflicted with disability in the form of rheumatism and chronic heart failure, and was obliged to retire. Following the installation of a communist government in Yugoslavia in 1945, his retirement continued, with an extra disability support to supplement his meagre income. He lived with his sister in Zagreb. He died on 10 March 1983, a totally anonymous person to the research community of Croatia. By the end of the 1980s, nobody at the School of Medicine even remembered his name. His name was rediscovered only during a scientometric investigation by Lackovićinto the history of the School of Medicine in the late 1980s. 11 It turned out that Sertićstarted his career in Lackovic's own department-the Department of Pharmacology, then part of the Institute for Pharmacology and Experimental Pathology (Pathophysiology).

INITIAL WORK IN ZAGREB
After completing his medical studies and internship at the Zagreb School of Medicine, Serticb riefly worked for the Ministry of Health. He obtained an academic position as a research fellow at the Institute for Pharmacology and Experimental Pathology (Pathophysiology) in 1926. Miroslav Mikulicǐć(Mikulichich, Miculichich), the head of the institute and a pupil of the Nobel laureate Otto Loewi, 12 entrusted Sertićwith the task of setting up an immunobiological ward. It was Mikulicǐc's idea to support a cross-disciplinary approach at the institute, where basic science and clinical wards were to be interconnected. 13 In this setting, Sertićhad the opportunity to pursue his interest in microbiology, which led him to his in vitro experiments with bacteriophages, by then a controversial topic with such prominent opponents to d'Herelle's ideas as Jules Bordet. 14 It was in these circumstances that Sertićpublished a paper in 1929 in which he described his experimental work done in Zagreb (figure 4). 15 In it, he explained the occurrence of bacteriolysins (alternatively labelled as phage lysins, endolysins or simply lysins): bacteriolytic enzymes produced by phages. Initial speculations on lysins being specific phagesecreted bacteriolytic enzymes and not degradation-associated bacterial by-products were published by d'Herelle and Eliava as early as 1921. 16   public waste waters, 19 and matched them to their phage database based on their developing classification principles. 20 As with developments in the area of transfusion medicine, 21 a uniform phage classification at the time represented a prevailing standard while also establishing the authority of d'Herelle's lab. Although a contemporary classification of viruses, including bacteriophages, has been developed by the International Committee on Taxonomy of Viruses (ICTV), original names assigned by Sertićare still used for many phage isolates; the best known example is wX174. 22 Since the institute was privately run and commercially strong, Sertic's isolates reached many research centres, and the names of strains were kept in use in accordance with his classification. One exemplary phage is wX174, first mentioned by Sertićand Boulgakov when describing their classification system. 23 The classification comprises a Greek letter, a Roman numeral and an Arabic number. The Greek letter w was used if the phage acted against multiple bacteria, whereas other Greek letters (τ, σ, δ, etc.) were used if the phage was specific to a single bacterium species (figure 5). The Roman numerals comprised 14 consecutive numbers, I-XIV. Thus, the correct reading of wX174 is phi ten 174 and not phi ex 174. Roman numerals were used to describe 'antigens' carried by the phage. These 'antigenic' reactions were based on the then already known methodology, but in this case specifically applied by Sertićand Boulgakov, according to which 'anti-sera' were produced by injecting a pure line culture of a given phage isolate into rabbits. 24 The rabbit serum was then used for crossneutralization tests with other isolates; the neutralization by a specific serum was one of several properties that they used to classify phages into groups designated by Roman numerals. The Arabic number was given in accordance to isolates from the private phage collection of d'Herelle's laboratory, and was therefore an arbitrary component. Sertićand Boulgakov's phage wX174 stands out for a number of reasons. Its DNA genome, the first example of a single-strand DNA as well as a covalent circular DNA, was the first one to be fully sequenced by Frederick Sanger's team, an achievement that garnered Sanger his second Nobel Prize, in 1980. 25 Ten years earlier, the 1959 Nobel laureate Arthur Kornberg successfully managed to synthesize in vitro a biologically active, exact copy of wX174 DNA, thus setting a milestone in the field of synthetic biology. 26 More recently, Craig Venter's group successfully assembled a strand of wX174 DNA by a full genome assembly method from synthetic oligonucleotides. 27 Notably, for every modern experiment in which wX174 has been used, it is possible to trace a direct line back to the initial encounter in 1950s between Boulgakov and Robert Sinsheimer, when the latter obtained the virus, originally Z. Lackovićand K. Toljan isolated and named by Sertićand Boulgakov. 28 As a scientist located in USA, Sinsheimer spread the virus throughout that country's laboratories and thence worldwide, reaching investigators such as Sanger, working in the UK. It is not an overstatement to claim that there is a bit of Sertićand Boulgakov's legacy knitted into all these famous experiments and into current work, where wX174 serves as a common model. When using the search terms 'wX174' and 'phi X 174' as descriptors in the PubMed database, we found 1859 publications (retrieved 14 July 2017); there were 267 000 hits in the less selective and less specific Google Scholar database.
Sertićdid not lose focus on his main duties of developing phage therapy, and started tackling the topic of bacteriolysins in more detail. 29 At the time, lysins were seen as a perfect tool for diagnosing specific bacterial strains. He and Boulgakov continued to perfect the isolation of various lysins and found them to be highly specific and sensitive for a specific strain, an improvement over any other method of detection at the time. After his initial 1929 paper, Sertićpublished eight more papers based on phage lysins. They include his final three publications (two co-authored with Boulgakov), printed in 1939, on the eve of World War II.

DEMISE OF D'HERELLE'S INSTITUTE
All of Europe was turned into a battlefield with the advent of World War II. Boulgakov, being a Yugoslav citizen, spent time as a prisoner in the Nazi internment camp near Campiègne in northern France. 30 During the summer of 1939, Sertićtook leave to spend time in Croatia. As a result of wartime exigencies in the Balkans, he was mobilized by the Yugoslav army in the spring of 1941, was briefly held captive and was then released by the Nazi occupiers back to the newly formed Independent State of Croatia. He was appointed as a professor and later Head of the Department for Bacteriology at the Zagreb School of Medicine in the autumn of 1941. From that period a handwritten and partially typed manuscript of what was intended to be a microbiology textbook has been retrieved from Sertic's papers. It consists of roughly 300 pages, describing the most up-to-date knowledge in bacteriology, virology and immunology, with almost a third of the manuscript being dedicated to the topic of bacteriophage.
In the events that brought chaos to Europe, d'Herelle's laboratory ceased to exist and, as others have documented, phage therapy essentially disappeared in the Western world. With antibiotics becoming the standard of care for treating infections, bacteriophages faded quickly from clinical practice only to blossom again as molecular biologists embraced phages as the central model for the gene; simple genomes, such as that of wX174, were crucial to much of the progress in basic research in the second half of the twentieth century.

SERTIC'S LEGACY: PHAGE AND LYSIN THERAPY TODAY
Evolving antibiotic resistance is almost universally viewed as an impending global health crisis. 31 By applying phages targeted specifically to individual patient isolates-the most common of

Forgotten Croatian scientist Vladimir Sertić575
which are E. coli and Pseudomonas aeruginosa-a better clinical outcome may be possible. Using the PubMed database as a source, we found a slow resurgence in the number of publications on therapeutic phages since the beginning of this century: in the year 2000 the total number of papers was 1050; this had risen to 1380 by 2016, with a yearly trend for a few of those being classified as clinical trials. Additionally, lysins have emerged as potential alternatives to antibiotics. During the second half of the twentieth century, some commercial kits for rapid bacteria detection contained lysins as part of the approach to enhance specificity. 32 Currently, lysins are seen as tools that may yield effective bactericidal therapies, and the scientific interest is considerable. A search on 25 October 2017, using the term 'lysin', returned 2256 publications in the PubMed database, 578 of which were published in or after 2011.
Based on Sertic's scientific bibliography, we can conclude that he was a prolific contributor to the phage research in the period before World War II. He published 35 papers in a ten-year period, most of those while working in d'Herelle's laboratory as the key person for research, development and devising of bacteriophage therapies destined for commercial use. A comprehensive bibliography of these publications, with the exception of those publications already cited in this paper, is provided in the appendix. Unfortunately, no peer-reviewed reports on actual therapeutic efficacy from that time are available. However, with the recent reconsiderations of phage or lysin therapy as a life-saving option when insurmountable antibiotic resistance is encountered, the role of the pioneers of bacteriophage research, including Vladimir Sertic, should not be forgotten.