Early Therapeutic and Prophylactic Uses of Bacteriophages Nina Chanishvili and Zemphira Alavidze Contents Introduction: A Brief History ... ................................................................. 2 Phage Therapy for Wound Treatment, Surgery, and Dermatology ... ........................... 6 Phage Therapy for Treatment of Enteric Infections ... .......................................... 10 Prophylactic Use of Phages ... ................................................................... 14 Intravenous Staphylococcal Bacteriophage: The Highest Achievement of the Georgian Scientists ... ....................................................................................... 16 Conclusions ... .................................................................................... 24 References ... ..................................................................................... 25 Abstract The long history of the discovery and therapeutic use of bacteriophages, espe- cially non-English-language journals, is often overlooked by modern researchers. While this body of evidence does not provide suitable proof of the safety and efficacy of phage therapy, it nonetheless demonstrates the potential for safety and efficacy and as such is worthy of attention by modern-day researchers in the field. Here, we discuss some of the early work carried out to develop clinical applica- tions of phage therapy for many diseases, with a particular focus on the huge amount of work carried out behind the Iron Curtain, in countries where phage therapy was (and in many cases still is) more commonly used than elsewhere in the world. N. Chanishvili (*) George Eliava Institute of Bacteriophage, Microbiology & Virology (EIBMV), Tbilisi, Georgia e-mail: [email protected] Z. Alavidze Phage Therapy Center, Tbilisi, Georgia e-mail: [email protected] © Springer Nature Switzerland AG 2020 1 D. Harper et al. (eds.), Bacteriophages, https://doi.org/10.1007/978-3-319-40598-8_12-1 2 N. Chanishvili and Z. Alavidze Introduction: A Brief History It has been argued that Ernest Hanbury Hankin may have been the first to publish on a bacteriophage-related phenomenon, when in 1896 he reported an agent which he demonstrated had antibacterial properties that could reduce titrers of the bacterium Vibrio cholerae in laboratory culture (Hankin 1896). The agent was found in the river Ganges in India, which was regarded by the population as a holy river as it was believed that its waters saved people from diseases (Adhya and Merrill 2006). Approximately at the same time, in 1989 Nikolai Fedorovich Gamaleya (a Ukrainian doctor bacteriologist-epidemiologist) published an article in the Russian Archives of Pathology, Clinical Medicine and Bacteriology (Gamaleya 1898), in which he described bacterial lysis of anthrax in distilled water after which an unknown agent was produced which in turn caused so-called transmissible lysis of other cultures of Bacillus anthracis. Other early observations of the phenomenon of bacterial lysis were made by Kruse and Pansini (cited from Kazarnovskaya 1933) who noticed that old pneumo- coccal cultures turned transparent due to death of bacterial cells. Later, Eijkman (1901) showed that an extinction of bacterial cells is not associated with exhaustion of media as it was believed before. Eijkman showed that after the death of one bacterial culture, it was not possible to cultivate another one, even of a related bacterial culture, on the same agar plate. According to Eijkman (1901), the extinc- tion of bacterial culture is a result of accumulation of toxic substances inhibiting bacterial growth and in some cases dissolving them. Condari and Kurpjuweit (cited from Kazarnovskaya 1933) found out that the inhibiting substance described by Eijkman (1901), if present in liquid media, was destroyed in a short period of time after heat treatment at 60–70 C. Long-term observations suggested that these sub- stances were produced by bacteria themselves due to their intracellular fermentative activities. They concluded that autolysis of bacterial cells occurs as a result of self- poisoning with the accumulated toxins. The active principle of this phenomenon was called autotoxin. The autotoxins caused lysis of old cultures; however, the autotoxins could not multiply like the d’Herelle phenomenon, and filtered autotoxins did not maintain an ability to lyse bacterial cultures (Kazarnovskaya 1933). Emmerlich and Low (1899) observed formation of an agglutinated mucous pellet of fresh bacterial culture in liquid media. This process was associated with increasing transparency of the media occurring within 2–3 days. Transmission of the transpar- ent media into a tube with growing culture caused lesser pellet formation, and the tube turned transparent. After 2–3 inoculations of the transparent media into the new culture, no more pellet developed. Emmerlich and Low explained this phenomenon by production of bacteriolytic enzymes (Emmerlich and Low 1899). In 1915 the English bacteriologist Frederick Twort (1915), who is recognized to be an original discoverer of bacteriophages, published in The Lancet his observa- tions about the destruction of the bacterial cells of Staphylococcus aureus and that the disrupted glassy areas might be transmitted to another culture and cause the same phenomenon. Twort (1915) believed that this phenomenon was caused by an Early Therapeutic and Prophylactic Uses of Bacteriophages 3 enzyme secreted by the bacteria and called the contagion “the bacteriolytic agent.” Twort’s work may have been ignored if Jules Bordet and Andre Gratia had not rediscovered his paper (Summers 2012). Two years after Twort’s publication, Felix d’Herelle published his article, Sur un microbe invisible antagoniste des bacilles dysentériques (d’Herelle 1917). D’Herelle started work at the Pasteur Institute in 1911, where he was engaged in the vaccine development and manufacturing process. In his spare time, he examined samples from dysentery patients. From the feces of several patients, he isolated an anti-Shiga “microbe” which was multiplied through many serial passages on its host bacterium and which could produce tiny clear circles on the lawn of the same Shigella culture. In 1917 he presented the results of his work to the French Academy of Sciences. Monitoring the patients with bacillary dysentery, d’Herelle discovered that shortly before the disappearance of blood in stool samples and recovery, some “agent” appears in the intestines, with an ability to dissolve the dysentery bacteria. In patients who have died of dysentery, the agent was never detected. This agent had the ability to reproduce itself only in presence of the host bacteria. D’Herelle gave the name to this agent –“bacteriophage” from Greek “bacteria eater.” He assumed that bacterio- phages were tiny creatures, much smaller than bacteria, with a corpuscular structure that parasitized bacteria and acted through production of specific enzymes. It is striking that these conclusions were made solely on the basis of d’Herelle’s empirical observations and intuition, since visualization of bacteriophage became possible only 22 years later (Summers 2012). The potential efficacy of phage preparations as antibacterial agents was demon- strated by tests on chickens using typhoid phages isolated from poultry. Due to the application of these phages, bird mortality was lowered from 95% to 5% (Kazarnovskaya 1933). As d’Herelle was sure that all types of bacteria had corresponding phages circulating in natural sources, the results of his animal exper- iments led to a decision to commence clinical trials to treat shigellosis at the Hôpital des Enfants-Malades in Paris under the clinical supervision of Professor Victor- Henri Hutinel, the hospital’s Chief of Pediatrics (Kazarnovskaya 1933; Summers 1999). To confirm bacteriophage safety prior to the experiment, the phage prepara- tion was ingested by d’Herelle, Hutinel, and some other hospital staff members – as was rather usual in those days. On the next day the bacteriophage preparation was given to a 12-year-old boy with severe dysentery. Even after a single administration, the disease symptoms were reduced, and complete recovery was achieved within a few days. Soon after this, three more patients diagnosed with bacterial dysentery were treated with one dose of the preparation, with improvement observed within 24 h after administration. D’Herelle, however, did not rush with publication of these results, so the first reported clinical application of phages belongs to Richard Bruynoghe and Joseph Maisin (1921), who used bacteriophages to treat skin infec- tions caused by Staphylococcus. Bacteriophages in that study were injected into and around surgically opened lesions. According to this publication, a regression of the infection occurred within 24–48 h. A number of promising studies followed (Rice 1930; Schless 1932; Stout 1933). Encouraged by these early results, d’Herelle and others continued studies of the therapeutic use of phages (e.g., d’Herelle used 4 N. Chanishvili and Z. Alavidze various phage preparations to treat thousands of people having cholera and/or bubonic plague in India) (Kazhal and Iftimovich 1968; Summers 1999, 2001). An enormous number of publications dedicated to discussions about the nature of bacteriophages were published in the 1930s and 1940s. Scientists were divided into two camps, supporting “precursor” and “viral” theories. In parallel with this, exper- iments on therapy and prophylaxis had been conducted by scientists