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, especially 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 applications 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]

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 pneumococcal 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 extinction 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 substances 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 transparent 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 observations 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 bacteriophages 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 demonstrated 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 experiments 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 preparation 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 infections 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, experiments on therapy and prophylaxis had been conducted by scientists working around the world. This information is impossible to embrace in one chapter. Therefore, we will discuss only a tiny number of publications illustrating results of phage treatment described in the early scientific literature. The discussion about the nature of bacteriophages continued for a long time until the invention of the transmission electron microscope by Max Knoll and Ernst Ruska, with the first phage images made in 1939 (Ruska 1940; Ackermann and Dubow 1987; Summers 2012). In 1933, Bayne-Jones and Sandholzen (1933) man- aged to film the bacterial burst caused by bacteriophage infection, albeit without coming to the right conclusion. According to the precursor theory, bacteriophages are developed endogenously; they exist in bacteria as a precursor which become lytic agents in the same way as trypsinogen is transformed into trypsin. This theory was supported by, among others, Gildemeister (1921), Bordet (1925), Northrop (1937, 1938), and Kruger and Scribner (1941). Another hypothesis was presented by Beckerich and Haudoroy (1922) who considered bacteriophages to be an external parasite which participated in the sexual cycle of bacteria. This theory was shared by the Russian scientists Jukov-Werezhnikov and Fruauf (1934), who considered that bacteriophages were a sexual (hereditary) non-visual form of bacteria. They assumed that bacteriophages fertilize bacteria, similarly as plant pollen fertilizes an ovule, during which phages force the bacterial cell to turn into an invisible stage. Another view was suggested by Gamaleya, who assumed that the bacteriophage phenomenon was associated with the production of a hormone “clustin.” He supposed that overproduction of clustin caused dissolution of the bacterial cell, turning it into a filterable form. The filtered clustin would cause dissolution again and again, over successive passages (cited from Jukov-Werezhnikov and Fruauf 1934). In 1934 a critical review of the available literature on phage therapy, based on 150 references, was published by the Journal of the American Medical Association, which drew conclusions not in favor of the therapy. It was directly stated that, “Experimental studies of lytic agent called ‘bacteriophage’ have not yet disclosed its nature. D’Herelle’s theory that this is a living virus parasite of bacteria has not been proved. On the contrary, the facts appear to indicate that the material is inanimate, possibly an enzyme” (Eaton and Bayne-Jones 1934). Such an assessment likely had a negative impact on investment in major research and production of bacteriophage, at least in the USA (Myelnikov 2018). This fact might have contrib- uted to d’Herelle’s decision to accept George Eliava’s invitation to conduct his research in Georgia. In the history of medicine, little is known about George Eliava, who was a colorful central figure in phage history. Obviously, the discovery of bacteriophages was inevitable; Eliava was one of those scientists who had observed spontaneous lysis of bacteria but could not explain this phenomenon. This is why he was strongly Early Therapeutic and Prophylactic Uses of Bacteriophages 5 interested in d’Herelle’s research. He supported d’Herelle’s theory about the viral nature of this phenomenon, along with his colleagues Asheshov, Bronfenbrenner, Gratia, Flu, Wollman, etc. Eliava first learned about d’Herelle and his discovery due to vigorous discussions held at the Pasteur Institute, where he had been sent by the Georgian government to advance his knowledge of bacteriology in 1919. Eliava spent several years working at the Pasteur Institute, where he met d’Herelle during one of his visits. This was the start of their collaboration and friendship. Eliava invited d’Herelle to Georgia, where they decided to found a World Center of Bacteriophage Research in that country. Without the support that Eliava provided to d’Herelle, much of the early knowledge of phage therapy may not have been achieved. Unfortunately, with his progressive thinking, tireless activities, and close collaboration with many foreign scientists, including d’Herelle, George Eliava became a victim of Stalin’s regime in 1937. He was pronounced to be a “people’s enemy” and was executed (Georgadze 1974; Chanishvili 2012a, 2012b). D’Herelle spent altogether 18 months during 1933 and 1934 in Georgia, collab- orating with Eliava and other Georgian colleagues (Chanishvili 2012a, 2012b). D’Herelle intended to move to Tbilisi permanently (a cottage built for his use still stands on the institute’s grounds); however, his intentions could not be realized. Nevertheless, the seeds of phage therapy found a good ground to grow and develop further in the former Soviet Union (Fig. 1).

Fig. 1 The ﬁrst conference on phage therapy in Tbilisi, Georgia, 1933–1934 (from left to right: unknown (sitting) Prof. Alexander Tsulukidze (standing), Felix d’Herelle, Prof. Simon Amiradjibi, George Eliava (all sitting), unknown (standing), Vladimir Antadze (standing)) 6 N. Chanishvili and Z. Alavidze

Phage Therapy for Wound Treatment, Surgery, and Dermatology

Soon after d’Herelle’s discovery of phages, many researchers around the world successfully isolated phages active against a wide range of bacteria, such as Salmo- nella Typhi (Alessandrini and Doria 1924), Shigella spp. (da Costa Cruz 1924), Streptococcus spp. (Dutton 1926), Corynebacterium diphtheriae (Fejgin 1925; Stone and Hobby 1934), Escherichia coli, Pseudomonas aeruginosa, Pasteurella multocida, Vibrio cholerae, Yersinia pestis (Krupp and Madhivanan 2015), and Neisseria meningitidis (Fruciano and Bourne 2007). The availability of these phages inspired doctors to develop speciﬁc treatments against different bacterial diseases. The phages were administered topically and systemically (orally and parenterally) (Rice 1930; Cipollaro and Scheplar 1933). Phage therapy was used experimentally to treat skin (Cipollaro and Scheplar 1933), eye (Town and Frisbee 1932), and bone infections caused by Staphylococcus (Albee 1933). Many doctors and researchers tried to use phages to treat suppurative wound infections using phage-soaked dressings. Over multiple articles, these doctors reported generally successful results (McKinley 1923, Rice 1930; Albee 1933); Albee and Patterson (1930) describe positive results using bacteriophage therapy to treat osteomyelitis, when after removal of infected bones and tissues they applied a plaster soaked with bacteriophages, which was changed several times during 7–9 weeks of treatment. Phage therapy of wounds was attempted during the Finnish Campaign in 1939– 1940. Early reviews of this work were published by Kokin (1941, 1946) and describe the use of mixtures of bacteriophages produced by the Eliava Institute of Bacteriophage, Microbiology and Virology, active against anaerobes, Staphylococ- cus and Streptococcus, for the treatment of gas gangrene. Phage mixtures were applied to 767 infected soldiers with a lethal outcome observed in 18.8% of cases compared to 42.2% in the control group. Using the same mixture of phages, other researchers reported a 19.2% lethal outcome in a group of soldiers compared to 54.2% of the control group (Lvov and Pasternak 1947 – cited from Krestovnikova 1947). Furthermore, this same phage mixture was used as an emergency treatment for wounds to prevent gas gangrene for soldiers in the Red Army both during and after World War II. Krestovnikova (1947) summarizes the observations of three mobile sanitary brigades carried out over periods of 2–6 weeks after evacuation to front-line hospitals. One brigade treated 2500 soldiers with phages. According to the reports, only 35 soldiers (1.4%) in this group showed symptoms of gas gangrene, while in the control group of 7918 wounded soldiers, 342 (4.3%) became infected. The second brigade applied phages to 941 soldiers, of which only 14 (1.4%) contracted gas gangrene, in contrast to 6.8% of soldiers in the control group. The third brigade treated 2584 soldiers, of these 0.7% developed symptoms of gas gangrene compared to 2.3% in the control group. Overall, the data described by these three independent brigades showed an average 30% decrease in the incidence of gas gangrene due to the use of a mixture of bacteriophages (Kokin 1941; Krestovnikova (1947). One of the pioneers in the application of phages in surgery was Alexander Petrovich Tsulukidze, a professor of medicine who began using such preparations Early Therapeutic and Prophylactic Uses of Bacteriophages 7

Fig. 2 Historical glass vials of bacteriophage suspensions as stored on the premises of the Eliava Institute, Tbilisi, Georgia in 1931 for the treatment of various diseases. According to Tsulukidze (1940, 1941), prior to initiation of treatment, the wounds were analyzed bacteriologically. Besides that, blood analyses were carried out before phage therapy and during surgical manipulations (bandages, puncture, etc.). The condition of the wound was thor- oughly described and temperature, pulse, breathing rate, etc. were all recorded. These examinations were also performed after each phage application. Initially the phage therapy was used only for the most severe cases where a lethal outcome was expected. Later, a wider group of patients was involved in the study. Since in the majority of cases bacteriological analysis indicated the presence of mixed bacterial infections (Tsulukidze 1940, 1941), Pyo-bacteriophage (a cocktail of phages used for the treatment of common pyogenic infections, active against S. aureus, Strepto- coccus, E. coli, P. aeruginosa and Proteus spp.) or a mixture of streptococcal and staphylococcal bacteriophages were applied for treatment (Fig. 2). The phage was administered topically or directly to the accessible part of the wound. Subcutaneous injection of phages was performed 3–4 times every second day to avoid the development of anti-phage antibodies. Additionally, phages were sprayed onto the top of wounds each time bandages were changed. All patients with injuries of soft tissues (38.3%) underwent “ordinary therapy,” which in this instance implied treatment with chloramines, rivanolum, and Vishnevsky ointment (all commonly used antiseptics at the time). These patients 8 N. Chanishvili and Z. Alavidze often had major tissue damage with penetrating or perforating wounds. The wounds were characterized by the accumulation of pus, infections, and surrounding inflammation, sometimes with necrotic foci, etc. A number of cases with an abscess/ phlegmon around a bullet or mine fragment wound underwent surgical cuts performed during first aid in a field hospital prior to the start of phage therapy. After purification of wounds with iodine solution and/or alcohol, followed by washing with 2% saline solution, the phages were sprayed on the top of the wounds. Simultaneously, 5–10 ml of phage (titer unknown) was injected remotely from the wound into the stomach wall, shoulder, or hip. The wound was bandaged with gauze soaked in phage. According to reports from that time, no cases treated with this method required additional cuts or any other surgery (Tsulukidze 1940, 1941). After a couple of phage applications, the body temperature usually normalized. To achieve a complete cure, only three to four procedures were required. Since the recovery from traumatic injuries and numerous lesions required an extended period, the wounds were stitched 6–8 days after phage treatment, so that further infection was unlikely. In general, phage therapy showed improvement within a number of days whereas standard therapy required several weeks (Tsulukidze 1940, 1941). Phage therapy was effective also for patients with bone injuries or open fractures. The wounds were purified with disinfecting solutions and washed with 2% saline before being sprayed with phages. Simultaneously, phages were injected intramus- cularly or subcutaneously at a site remote from the wound. Following this, a plaster cast was applied. The treatment with phages resulted in a faster reduction of pain, improvement in patients’ general condition, and healing of wounds beginning after 2 or 3 days (Tsulukidze 1940, 1941, 1942a, 1942b; Arshba 1942; Makashvili et al. 1942; Sirbiladze 1942; Pokrovskaya 1942). It was noted also that by using phage therapy prior to plastering, it became possible to avoid moistening the plaster, which usually necessitated its replacement and could lead to the development of secondary infections. The plaster could remain unchanged for up to 60 days (Tsulukidze 1940, 1941, 1942a, 1942b). Importantly, in cases of severe hip, shin, forearm, and shoulder injuries, which normally require amputation, application of phage therapy avoided amputation if wounds were left for 10–30 days in blind cast plasters. Very often, phages against the main causes of wound infections were isolated from patients that had never received phage therapy before (Tsulukidze 1942a, 1942b). Phage-treated wounds were examined several times before and during the application of phage therapy. These examinations showed resulting complete sterilization of wounds and in cases of clinical improvement a loss of virulence by the pathogen (Tsulukidze 1940, 1941, 1942a, 1942b). Sirbiladze (1942) described morphological changes of clostridial colonies plated on agar. Before phage therapy the colonies of Clostridium perfringens formed entire, round colonies, while the isolates obtained from the same wounds after the treatment formed colonies with ragged edges. Besides that, animals infected with these strains of C. perfringens survived, implying that their toxicity and pathogenic properties were significantly weakened (Tsulukidze 1942b, Arshba 1942; Makashvili et al. 1942). Successful wound treatment with bacteriophages during the Finnish Campaign and World War II was also reported (Pokrovskaya 1942; Fedorovich 1944; Sutin 1947; Fisher 1949). Early Therapeutic and Prophylactic Uses of Bacteriophages 9

Phage therapy was applied in different fields of medicine, such as stomatology (Ruchko and Tretyak 1936), ophthalmology (Rodigina 1938), urology (Tsulukidze 1957), and gynecology (Purtseladze 1941), among others. The results of phage therapy in dermatology are especially important. The successful treatment of deep forms of dermatitis caused by S. aureus with specific bacteriophages has been described in a number of articles (Beridze 1938; Vartapetov 1941, 1947, 1957; Izashvili 1940, 1957; Khuskivadze 1954; Gvazava 1957; Shvelidze (1970); Vartapetov et al. 1974). The oldest study in the field was carried out by Beridze (1938). The author described 143 cases of purulent skin infection caused by S. aureus divided into 2 major groups exhibiting either deep or superficial forms of the disease. The group with deep infections included 90 patients with furunculosis (multiple boils) (73 cases), abscesses (10), and hidradenitis (7). A group of 53 patients with superficial skin infections included cases of impetigo vulgaris (29), impetigo contagiosa (13), and various other diagnoses (11). The patients’ ages ranged between 1 and 60 years, with the majority being workers between 20 and 35 years of age. The duration of the illness in patients with acute forms varied from 1 to 7 days, whereas the chronic forms lasted from several weeks to 3 years. The methodology of treatment was described in detail. Initially the area around the infected site was cleaned with a disinfectant solution, moving from the periphery to the center. The pus was then released from the infected area in order to decrease the bacterial load and allow access for the bacteriophages. Simultaneously, swabs were taken to isolate the infecting bacteria and assess their susceptibility to phages. An initial dose (0.5 ml) of bacteriophage preparation was injected directly into the wound and surrounding healthy tissue. The wound was then covered with phage and bandaged. If, on the following day, there was no evidence of irritation, swelling, or any allergic reaction, treatment with phage was continued, with the dose of injected phage gradually increased up to 1 ml on the second day, 2 ml on the third day, and so on. The doctors decided whether to increase the dose each day based on the appearance of the wound and skin reaction. Altogether, 4–5 phage injections were given. After this course, the patients were switched to so-called indifferent therapy which included zinc salve or similar medication. After 3 days the patients were examined again. If the infection persisted, phage therapy was continued in the form of applications onto the previously disinfected wound. If no effect was observed, the patients underwent phototherapy. The patients underwent repeated medical examination after 3 weeks, 3 months, and 10 months. In this group of 143 patients, 108 (75.5%) were successfully treated and an improvement was seen in 11 cases (7.7%). No effect was observed in 18 cases (12.6%), and the fate of 7 patients (4.9%) was unknown (Beridze 1938). In his paper, Vartapetov (1957) listed numerous authors studying the clinical efficiency of bacteriophages (staphylococcal and streptococcal phages and Pyo-bacteriophage) for the treatment of furuncles, carbuncles, hidradenitis, abscesses, and so on. Vartapetov (1957) summarized the data of over 6000 patients involved in phage therapy studies, demonstrating that in all cases healing occurred within 4–8 days. Clinical success rates ranged from 70% to 100%. In general, the 10 N. Chanishvili and Z. Alavidze best results were seen in cases of treatment of abscesses and sycosis caused by staphylococcal infection. An interesting study was performed by Shvelidze (1970) on 161 patients with chronic and frequently relapsing infections. Sixty-two patients were diagnosed with furuncles (boils) and furunculosis, 54 with carbuncles, and 45 with hidradenitis. Despite antibiotic treatment, some patients suffered with chronic infections, in some cases for as long as 20 years. The patients complained of fevers, headaches, weakness, insomnia, and/or movement difficulties. Bacteriological analyses showed that in 82.7% cases the infection was caused by coagulase-positive penicillin- resistant Staphylococcus aureus. Phages were administered topically via intradermal injections performed every second day. The phage was administered to patients in increasing doses ranging from 0.1 to 0.5 mL. In total, 7–10 injections were given around the infected site. It is important to underline that the results of phage therapy were compared with the previously performed antibiotic treatment, which was considered as a control. Successful results were achieved in 94.4% (152 cases), 4.3% (7 patients) showed a significant improvement, and only in 1.3% of cases (2 patients) was no improvement observed. The patients were monitored for a 4-year period following treatment. Relapse was observed in 8.5% of cases; however the severity of these subsequent infections was of relatively minor concern, and an additional course of phage therapy resulted in complete cure (Shvelidze 1970). Many authors (Beridze 1938; Gvazava 1957; Vartapetov 1957; Shvelidze 1970; Vartapetov et al. 1974) drew attention to the immunostimulation potential of staphylococcal bacteriophages. Based on their observations, the authors (Beridze 1938; Gvazava 1957; Vartapetov 1957) suggested the following:

1. Phages kill (lyse) the appropriate host bacteria (direct effect of phage therapy). 2. The killed bacteria are present in the bloodstream as pieces of bacterial cell wall and debris (i.e., antigens) which stimulate the immune system. These antigens might trigger the immune system in a manner that could be more diverse and better presented than recombinant vaccines and better folded than other forms of inactivated vaccines, such as heat-killed pathogens (indirect action of phage therapy).

Phage Therapy for Treatment of Enteric Infections

In the 1920s and 1940s, intestinal infections caused by Salmonella and Shigella species were a huge problem all over the world (Beckerich and Haudoroy 1922; Alessandrini and Doria 1924, da Costa Cruz 1924; Rolleston 1926; Compton 1929; Karamov 1938; Karpov 1946). Karpov (1946) provided epidemiological data on mortality rates at different times and at various geographic locations. Mortality rates in cases of typhoid fever varied between 7 and 10%. In Baku (Azerbaijan) in 1932, the mortality rate was 5.8%. Similarly, in one of the main hospitals in Leningrad (now St. Petersburg, Russia), the mortality rate in 1931 also attained 5.8%. During an outbreak in Rostov (Russia), in 1926, mortality rates reached 8.2% (Karpov Early Therapeutic and Prophylactic Uses of Bacteriophages 11

1946). In autumn of 1926, an outbreak of typhoid fever started in Hanover, Ger- many, where 4220 cases were reported, among which 320 (7.6%) lethal outcomes were recorded. A water supply system was recognized to be a main source of this outbreak (Rolleston 1926; Speigel Online 2011). These figures indicated the urgent need to introduce novel therapeutic means to combat these infections. The first trial to treat human intestinal infections was performed by d’Herelle in 1919 and included only four patients suffering with dysentery. The phage treatment was administered as a single dose, and the symptoms of dysentery disappeared by the next day (d’Herelle 1917). After d’Herelle’s experiment, many scientists started to use phage therapy to treat dysentery, however with varying success (Davison 1922; Spence and McKinley 1924; Compton 1929; Riding 1930; Querangal des Essarts 1933; Kessel and Rose 1933; Haler 1938; Johnston et al. 1933; Karamov 1938; Karpov 1946). For example, Davison (1922) described 12 cases of bacillary dysentery caused by Shigella flexneri, among which 7 patients received phages orally, while 5 were treated by enema. Only 5 out of 12 patients (42%) overcame the infection. Failure in the other cases was explained by the fact that the therapy began too late when the disease was already in its peak (19). According to da Costa Cruz (1924), phage therapy was the best treatment for bacillary dysentery, with the positive results achieved in 24–48 h. These statements, however, were not supported with reliable statistics. One of the most detailed early reports belongs to Compton (1929), who described the treatment of dysentery in the city of Alexandria, Egypt, performed in 1927–1928 with the cooperation of the doctors willing to evaluate the effects of phage therapy in the treatment of dysentery. The polyvalent phage lysate included phages active against S. shiga, S. flexneri, S. hiss, S. sonnei, and S. gay. Only those patients whose diagnosis was confirmed etiologically were included in the study. Each patient received three ampoules containing 2 mL of phage lysate. The patients were provided with instructions on the use of the phage and a questionnaire. In 1927, almost 50 patients were treated and about 150 in 1928. Among 200 patients, 92 did not complete the treatment and returned the ampoules. Of these, only 66 were intact and applicable for subsequent use. Therefore, the authors assumed that 108 cases successfully completed the cure since they did not return for additional visits to their doctors. Thus, coming out of this assumption, Compton (1929) concluded that the cure rate was 108/200 (54%). The remaining ampoules Compton used for treatment of 66 patients diagnosed with dysentery. For this experiment, the author developed a semi-qualitative method of evaluating the recovery of the patients, scoring the success results as “very good” (35 cases), “good” (10 cases), “moderately good” (6 cases), “partial failure” (5 cases), and “failure” (10 cases). Four out of ten negative cases Compton removed from the study, since their treatment started too late when the patients were already severely sick. Thus, summarizing very good and good results, the total success rate achieved was indicated as 72.6% (45/62) (Compton 1929). Due to Compton’s(1929) study, it became possible to analyze in detail the reasons for phage therapy failures. Analysis of the results revealed that the age of the patient, the duration of illness prior to phage treatment, and resident bacteria other than the targeted species were important factors influencing the outcome of the phage treatment. In particular, it was observed that phage treatment was the least 12 N. Chanishvili and Z. Alavidze successful with children under 1 year old, with the success rate increasing in direct proportion to age. Early start of phage therapy treatment, however, was critical to a successful outcome. It was shown that if the patient had been ill for 3 or fewer days prior to treatment, then the success rate could be as high as 90%. The longer the duration of the period between the onset of illness and start of phage therapy, the lower was the success rate. Later studies conducted by Riding (1930) and others (Querangal des Essarts 1933; Kessel and Rose 1933; Johnston et al. 1933; Haler 1938; Murray 1938; Goodridge 2013) appeared to be less informative, since no information was reported regarding the doses, duration, and the number of times the phage preparation was administered, control groups (Johnston et al. 1933; Murray 1938). The failures of phage therapy sometimes were caused by the fact that no preliminary in vitro susceptibility tests were done prior to clinical trials (Murray 1938). Collectively, these reports reveal much diversity in results and conclusions. Comparisons of the studies are impossible due to lack of information regarding concentration of phage, numbers of different phages employed, method of preparation, method of administration, and the fact that in many of the reports no controls were included. The largest clinical study of therapeutic anti-dysenteric phages was reported by Sapir (1939) who describes a total of 1064 cases of dysentery treated with bacteriophages in two different Moscow clinics. The patient group included 767 men and 297 women ranging in age from newborns to 79 years old. Dysentery was diagnosed using bacteriological tests (362 patients), clinical observations (512 cases), and clinical colitis (190). Bacteriological analysis of 362 patients indicated that dysentery in 289 cases was caused by Shigella dysenteriae type 1 (22 resulting in a lethal outcome), 69 cases of S. flexneri, and 4 cases of S. dysenteriae type 2. A standard phage therapy, using the dysenteric bacteriophage preparation that was developed by the Mechnikov Institute in Moscow, was applied to every age group as described below. The exact content of this preparation has not been specified. A daily dose of phage for an adult was 20 ml and 10 ml for a child (titer 109 to 1011 using the Appelmans method of serial dilutions in bacteriology media which does not allow to quantify phages in the stock solution) (Appelmans 1921; Chanishvili 2012a; Rohde et al. 2018). The dose was divided into two portions and given to patients at midnight on the day of arrival and at 4 am to minimize the inactivation of phage by any meal residues. The comparable time of phage administration to all patients facilitated the evaluation of the results of the phage therapy. The patients were given a magnesium-soda solution (magnesium 10 g/1 L + sodium bicarbonate 20 g/L), initially 6 h prior to phage therapy and then every 2 h for the following 12 h. Adults were given 100 ml of the solution per dose, and children were given 10–50 ml depending on their age. The solution was given with the aim of providing optimal conditions for phage propagation and also to help clear the intestines. The patients were kept on a strict diet during the first 48 h. The author concluded that the application of this dose of phage divided into two portions and administered over the course of a single day was sufficient and did not need to be repeated (Sapir 1939). Early Therapeutic and Prophylactic Uses of Bacteriophages 13

According to Sapir (1939) the application of phage therapy significantly decreased the duration of hospital stays (11–20 days), in contrast with symptomatic treatment (43 days) or even with specific (serological) treatment (22 days). The paper highlighted that early use of phage therapy reduced hospitalization time and that usually after 1–2 days of phage therapy a dramatic improvement in the patient’s condition was observed, as evidenced by less frequent and less watery stools with less blood and/or mucus. Sapir (1939) reported also that after one day of phage treatment, the number of patients with bloody stools decreased from 100 to 74, and on the fifth day of treatment, only 4 patients remained with this symptom. A single week of phage therapy resulted in reduction of symptoms such that 95% of patients could be released from hospital. Nevertheless, a lethal outcome was observed in 47 cases (4.4%), although it was noted that these patients suffered with dysenteric pancolitis and other severe degenerative changes of the parenchyma of various organs, as well as colonic ulcers, etc., which are typical for long-term infection, as verified by the postmortem studies. The author concluded that phage preparations should be given to every patient showing symptoms of dysentery, independently of whether the patient is arriving at hospital, being seen by ambulance or asking for medical help at home. This measure would have not only therapeutic but prophylactic effect as well (Sapir 1939). Lipkin and Nikolskaya (1940) performed phage therapy on 100 patients suffering from dysentery. A control group of 50 patients received ordinary medication, such as purgative salts, which were used in most cases. In 21 cases the patients underwent serum therapy. In five severe cases, a combined phage and serum therapy was used. All patients were maintained under the same conditions, in terms of care, diet, etc. Phages produced by the Tbilisi Institute of Vaccine and Sera and the Kuibishev Institute of Epidemiology and Microbiology were used in these studies (An old name of the Eliava Institute of Bacteriophage, Microbiology & Virology). The titers of these phage preparations were 109 to 1011 by the Appelmans method (Appelmans 1921; Rohde et al. 2018). Five ml of phage was given to patients orally together with 2% soda solution 3 times per day. After receiving the phage, the patients fasted all day. In almost every case the phage treatment was performed for one day. Only in 6 cases was the phage treatment at the same dose performed over 2 days. The majority of patients (66%) received the phage within the first 5 days of the start of infection. The development of the disease was evaluated through observations of stool frequency, presence of mucus, blood, cramps, etc. Lipkin and Nikolskaya (1940) reported a significant effect of phage therapy even in cases where the treatment was started rather late. 25% of patients (n ¼ 100) stopped reporting painful symptoms by the second day of treatment. 79% did not show pathological symptoms by the fourth day, and 100% did not by the sixth day, after which stool had normalized. These data are in contrast to the results obtained with standard therapy, where only 2% (1 case out of 50 patients) showed an improvement on the second day of treatment, 14% on the fourth, and 46% on the sixth day. It is noteworthy that patients with more mild symptoms were included in the control group, while those with relatively severe illnesses were included into the experimental group. According to the authors, the fact that these patients showed an improvement as 14 N. Chanishvili and Z. Alavidze soon as they got phage treatment illustrated the effectiveness of this method. Relief of symptoms in patients treated with serum therapy was recorded in 33% of cases (7/21) on the fourth day of treatment and in 67% on the sixth day, indicating that serum therapy resulted in the slower relief of symptoms than phage therapy. Five patients subjected to serum therapy remained sick over 10 days. These patients later underwent successful phage therapy (without further serum therapy) as well. Phage preparations were generally considered to be particularly efficient for the treatment of intestinal infections (Podvarko 1964). Vlasov and Artemenko (1946) described the results of treating 30 chronic dysentery patients with phages. Many of the patients were exhausted by infection and were bedridden. A dry tablet preparation known as “phage-vaccine”–a combined preparation comprising, after recon- stitution in saline, 106 killed cells/ml and 107 (by Appelmans 1921) – was used. The patients had suffered with infections for 1–2 years, and in 70% of cases rectoscopic investigation indicated the presence of bleeding ulcers. Prior to combined phage-vaccine therapy, all of the patients underwent multiple courses (1– 8 times) of therapy with antibiotics and sulfonamide preparations. After the phage- vaccine therapy, the authors reported curing 26 patients (86.7%) within 10–20 days. Assessment of the results was based on improvements in the general condition of patients including normalization of stools and recovery of the mucous layer of sigmoid colon and rectum (Vlasov and Artemenko 1946).

Prophylactic Use of Phages

Phages have been used extensively in the former Soviet Union for prophylaxis in regions with a high incidence of infections and also in communities where rapid spread of infections might occur, such as kindergartens, schools, military accommo- dation, etc. (d’Herelle 1935, Belikova 1941, Blankov 1941, Blankov and Zherebtsov 1941, Kagan et al. 1964, Florova and Cherkass 1965, Agafonov et al. 1984, Anpilov and Prokudin 1984, Chanishvili 2012a, 2012b). The application of phages for prophylaxis was carried out in 1929–1930 against the diseases that were the most severe and important at that time, such as dysentery, typhoid fever, staphylococcal infections, etc. The first mass application of dysenteric bacteriophages in the USSR was performed in Alchevsk (Donbas region) in Ukraine in 1930 (Ruchevski, “Vrachebnaya gazeta,” 1931, 21: 1586, cited by Krestovnikova 1947). An experiment on the prophylactic use of phages was later successfully carried out in 1935 on thousands of people in regions with a high incidence of dysentery (Melnik et al. 1935, Belikova 1941, Blankov 1941, Blankov and Zherebtsov 1941, Vlasov and Artemenko 1946). The results were reported at scientific conferences in 1934 and 1936 in Kharkov and in 1939 in Moscow, after which the dysenterial phage preparation was finally approved as a preventive measure for mass application (Krestovnikova 1947). According to Krestovnikova (1947), it was recommended that repeated seasonal prophylactic “phaging” be carried out in areas where dysentery was endemic. Later modifications included Early Therapeutic and Prophylactic Uses of Bacteriophages 15 formulating the dysenterial phages as dry tablets, which also began to be included in clinical studies. One of the most dramatic examples of prophylactic use of cholera bacteriophages is related to the Stalingrad battle and an outbreak of cholera with many lethal outcomes in 1942 and 1943. The prominent Soviet bacteriologist Yermolieva, with her staff, organized bacteriophage production in the city. Doses of cholera phage were given to 50 thousand people every day during 5 consequent days (bread was given only after “phaging”). The outbreak subsequently ceased (Yermolieva 1939, 1942; Yermolieva and Yakobson 1943, 1949). During the monitoring period, lasting 3 months, the presence of Vibrio cholerae in the feces of convalescents was not registered (Yermolieva and Yakobson 1949). Babalova et al. (1968) describe the results of prophylactic measures carried out in 1963–1964 using phage tablets with an acid-resistant coating. Over 30,000 children from the age of 6 months to 7 years were involved in the study. Prophylactic phage treatment was carried out on children living on one side of the street, while those living on the other side did not get the phage treatment and thus were considered a control group. The phage tablets were administered either before or 2 h after meals. Children from 6 months to 5 years received 1 tablet (equal to 20 ml of phage). The titers of each component of this polyvalent dysenteric preparation was equal to 104–105 by Appelmans (1921), while children over 5 years received 2 tablets. The effect of phage prophylaxis was evaluated on the basis of clinical symptoms and in some cases also based on bacteriological analysis. The incidence of acute dysentery in the control group was 3.8 times higher than in the experimental group (Babalova et al. 1968). Later studies on mass prophylaxis of intestinal diseases by the application of phages were performed in Red Army units by military doctors. For prevention of dysentery and typhoid, epidemic strain-specific phages were also used, with two tablets administered once every 5–7 days during the outbreak season. The authors reported about six- to eightfold decrease of incidences of intestinal infections in the test groups in comparison with the controls (Florova and Cherkass 1965; Agafonov et al. 1984; Anpilov and Prokudin 1984; Kurochka et al. 1987; Chanishvili 2012a, 2012b). Sayamov (1963) reports the results of therapeutic and prophylactic trials using an anti-cholera bacteriophage preparation, performed by Soviet doctors in East Pakistan in 1958 and in Afghanistan in 1960. During a cholera outbreak in Dacca (East Pakistan) in 1958, only 22 patients with severe conditions underwent phage therapy treatment. Each patient was given a single intravenous dose of phage suspension (5– 10 ml) prepared on saline solution and simultaneously an oral dose of phage suspension (30 ml), for three consecutive days (phage titer is unknown). Only two lethal outcomes were registered, in contrast with the fatality rate at the Kandahar hospital, which appeared to be about 50%. At the same time, the Soviet doctors used phage therapy prophylactically in East Pakistan. It was reported that phage prophylaxis was performed on a large group of around 30,000 people. These measures successfully ceased spread of cholera epidemics, since no cases were reported in these areas (Sayamov 1963). 16 N. Chanishvili and Z. Alavidze

Phage prophylaxis was implemented in Afghanistan in 1960, which included patients admitted to the hospital as well as healthy hospital staff (total >1600 persons). The doses and duration of the treatment were the same as described above. Only 4 lethal outcomes out of 119 cholera patients (3.5%) were registered as a result of these measures. No complications were observed as a result of phage therapy. Similar prophylactic measures were reported also in Kataghan Province in the north of Afghanistan in 1960 (Plankina et al. 1961; Sayamov 1963). In this case the majority of the patients were treated at home (about 90%) because of the difﬁculties associated with transportation of the patients to the hospitals and limited number of bed places. Like in other cases, the patients received a dose of 20–30 ml (or, in particularly severe cases, as much as 50 ml) of phage suspension, accom- plished with one or two intramuscular injections (intravenous administration was not used). In addition to phage therapy, a single dose of cholera vaccine was used for preventive purposes. This complex treatment was applied to a healthy part of each village’s population. Sayamov (1963) and Plankina et al. (1961) underlined the role of phage prophylaxis in prevention of new cholera cases in rural populations as the best success was observed only in those villages where the whole population was subjected to the phage treatment. Thus, altogether approximately 270,000 persons in all regions underwent phage treatment. The information about the control groups is missing in this publication which makes it difﬁcult to evaluate the contribution of phages and of the vaccine in the observed prophylactic effect. However, Sayamov tended to conclude that the effect of anti-cholera prophylactic largely was dependent on bacteriophage treatment. He suggested that phage prophylaxis may be used as an important supplement to the standard measures for cholera control (Plankina et al. 1961; Sayamov 1963).

Intravenous Staphylococcal Bacteriophage: The Highest Achievement of the Georgian Scientists

The story of phage therapy would be incomplete if we did not mention the development of intravenous anti-staphylococcal phage and its use in humans. Attempts to produce intravenous bacteriophage preparations were started by d’Herelle using crude yeast extract medium phage lysates (Antadze 1957). These experiments continued into the early 1930s in the Soviet Union, and the earliest study dedicated to this to be found in the library of the Eliava Institute was published by Ebert and Shapiro (1938). In this article the authors briefly describe previous animal trials on rats and rabbits, which were performed by intravenous infection of the animals followed by a single intravenous administration of phage. No protective effect against infection was apparent, and the animals died. This was in contrast to the positive impact of topical bacteriophage therapy, as observed by other doctors. Therefore, the experiments of intravenous administration of phages continued. Skvirskyi et al. (1938) used combined intravenous and intramuscular administration of bacteriophage to treat typhoid fever. These authors were among the first to administer phage therapy intravenously (1–2 ml) against typhoid fever. Skvrskyi Early Therapeutic and Prophylactic Uses of Bacteriophages 17 et al. (1938) observed a lowering of temperature but did not attribute this to the use of phage. In 1930 Ruchko and Melnik (cited by Krestovnikova 1947) reported experiments performed on 69 patients suffering with typhoid fever. Like previous authors, they also used an intravenous mode of administration and also observed a lowering of temperature and a shortening of the duration of illness. However, many authors reported a rise in temperature of 1–2 C prior to lowering (Krestovnikova 1947). During World War II, Yermolieva, in severe cases of cholera, used bacteriophages intravenously. 5 ml of phage lysate diluted in 2 L of saline solution was slowly introduced into the vein twice, with a two-day interval between treatments. The treatment continued orally using the following regimen: 30 ml of cholera phage divided into two doses consisting of 15 ml of phage diluted in 20 ml of boiled (sterile) water administered 3 h apart. The treatment continued for 3 days (Yermolieva 1939; Yermolieva 1942; Yermolieva and Yakobson 1943, 1949). These measures decreased the number of cholera incidences and helped to stop the spread of the epidemic among the civilian population and army units as well. Positive results of intravenous administration of phage therapy among patients with acute septicemia caused by anaerobic infections were described by Arsentieva (1941). The phage was administered as transfusions in doses of 50–100 ml with intervals of 2–3 days. The author refers to shivering and slight rise of temperature which was treated with caffeine and warming of the patient (Arsentieva 1941). Kokin (1941, 1946) used intravenous administration of phages in cases of staphylococcal infections. 30 ml of specific anti-staphylococcal bacteriophage diluted in 300 ml of saline solution was used for transfusions. An intensive reaction following intravenous administration was explained by release of bacterial toxins and activation of phagocytosis. In cases of moderate body reaction, the dose of phage was increased up to 50–60 ml in 300 ml of saline solution (Kokin 1941, 1946). Yukelis (1946) reported a 72% success rate of phage therapy in cases of furunculosis and deep ulcerous pyodermitis using intravenous administration of staphylococcal bacteriophages. The phage was prepared on protein-free bacteriology media, and 10 ml was administered every second day. The author did not observe any side effects and recommended performing intravenous transfusions of 40–50 ml every day (Yukelis 1946). A very interesting study was performed by Manolov et al. (1948) who reported on the intravenous application of bacteriophages for treatment of typhoid fever. The low efficacy of the therapeutic effect after oral administration of bacteriophages led the authors to conclude that intravenous administration could be used. For treatment of typhoid fever, the authors applied 20–25 ml of bacteriophage prepared in saline solution containing minimal amounts of organic contaminants. Safety of this bacteriophage had been proven in experiments on rabbits and white mice. It was shown that intravenous administration of the typhoid bacteriophage in animal models protected the mice from the development of infection when challenged by a dose of the typhoid culture (Manolov et al. 1948). 15–20 min after administration of the intravenous phage, patients complained of shivering. After 2–3 h a rise in temperature was observed which in some cases was followed by a feeling of nausea and often vomiting. 12–14 h after the phage injection, the temperature was normalized. 18 N. Chanishvili and Z. Alavidze

After 24 h, however, it rose again to the same level as seen after 2–3 h. As a result of this, the authors applied several phage injections every day or every other day. Unfortunately, the number of patients is not indicated in the study, but the results led to the following conclusions:

1. Oral administration of bacteriophage was regarded as unsuitable due to the speciﬁcity of pathogenesis of typhoid fever. And low doses (2–10 ml) of typhoid bacteriophages administered orally were inefﬁcient. 2. Intravenous use of 20–25 ml of typhoid bacteriophage, prepared in saline solution, every day over 3 days led to a decrease of temperature and shortening of the fever period, improvement of the general condition, and complete cure (Manolov et al. 1948).

Although phages were administered intravenously in early studies during the 1930s and 1940s, this type of therapy was rejected due to the unpleasant side effects, including a rise in temperature up to 39C, shivering, headaches, etc. The negative reactions usually occurred for 40–60 min following treatment. However, no lethal outcomes were reported (Skvirskyi et al. 1938; Yukelis 1946; Kokin 1941; Kokin 1946; Krestovnikova 1947; Manolov et al. 1948; Antadze 1957). The Eliava Institute of Bacteriophage has had particular success in the elaboration of intravenous bacteriophage preparations for the treatment of S. aureus septicemia. Antadze (1957) wrote that the organic residuals emerging during manufacturing of the phage preparations create difficulties for their parenteral use, since they might cause a number of side effects due to their sensitizing impact on the body’s immune system. Taking into consideration the fact that during the manufacturing process (i.e., at the stage of the growth of bacteriophages in the bacteriology media), the host bacteria are unable to completely utilize the substrate, researchers from the Eliava Institute decided to exclude the unessential protein fragments of the media from the phage propagation process. They determined the essential portion of bacteriology media which would be necessary for multiplication of the host bacteria and ampli- fication of the phage particles to allow them to reach sufficient titer. This goal was achieved, and the Eliava staff succeeded in propagating the bacteriophages on media diluted with a saline solution so that they would reach titers of 108–1010 by Appelmans (1921). Successful preparation of anti-pyrogenic staphylococcal bacteriophage for parenteral use was achieved in the 1970s. These studies, including manufacturing of the intravenous staphylococcal bacteriophage preparation and its use in animal and clinical trials, were performed under the leadership of Professor Teimuraz Chanishvili. Research to elaborate a protein-free apyrogenic media suitable for the manufacture of intravenous staphylococcal bacteriophages was carried out simultaneously at the Tbilisi Institute of Vaccine and Sera (Chirakadze and Chanishvili 1964; Chanishvili et al. 1974; Nadiradze 1983) and the Gorky Institute of Epidemi- ology and Microbiology (Anikina 1982). Anikina (1982) tried to reproduce staphylococcal septicemia in an experimental animal mouse model and carried out a total of 30 experiments on 750 animals. Early Therapeutic and Prophylactic Uses of Bacteriophages 19

Further studies were performed in rabbit models using a generalized staphylococcal infection in rabbits with three S. aureus strains. Each strain induced infectious processes of different severity and duration. The impact of the intravenous bacteriophage was studied in acute and chronic septic models. The rabbits were given phages intravenously at 5 ml/kg, and injections were repeated three times over one day. The treatment did not prevent lethal outcomes but that occurred on the 18th day, much later than in the control group, and kidney abscesses were not seen. The animals in the control group died between the fourth and tenth days. The next series of experiments was performed using a chronic septicemia rabbit model using two phage preparations:

1. An apyrogenic staphylococcal phage preparation made by the Tbilisi Institute of Vaccine and Sera, prepared on synthetic media with the addition of yeast extract 2. An experimental staphylococcal phage preparation made by the Gorky Institute of Microbiology and Epidemiology, manufactured on media containing amino- chlorine hydrolysate

Altogether, 35 animals were divided into 2 groups of 10, with 15 left as a control. The animals were infected with the strain S. aureus #79(4 106 cfu/kg). A day after infection the rabbits were given 3 ml/kg of the different phage preparations for 5 days. The animals from both experimental groups were cured within 7 days (Anikina 1982). Both preparations appeared to be almost equally effective. In the next experiment, rabbits received combined phage and antibiotic (penicillin) therapies. The animals were infected with the penicillin-resistant strain, S. aureus #36(2 106 cfu/kg), which caused prolonged generalized infection. The phage was given intravenously 3–5 ml/kg. Treatment started on the second day after the infection and continued for 5 days. Penicillin was administered intramus- cularly, first 4 h after infection (200,000 units per 1 kg of weight) and continued for 5 days. The animals were observed for 14 days, during which bacteriological analyses were performed on the first, second, third, fourth, fifth seventh, and ninth days. After 14 days of observation, all animals from the experimental and control groups were euthanized and their internal organs checked for bacterial loads. Neither of the 5-day treatments performed separately with the phage or antibiotics resulted in eradication of infection. The best results were achieved after the combined treatment with phage and penicillin: Only in one case was S. aureus detected, 3 days after infection. However, dissection of this animal did not show any pathological changes (Anikina 1982). Since high doses of antibiotics may cause various side effects, the author decided to perform the same experiment using low doses of penicillin (25,000 units/kg). 15 rabbits were divided into 3 groups. The first was an untreated control group; the second group of rabbits was treated with injections of 25,000 units of penicillin per kg, with the antibiotic given 4 h after infection and then intramuscular injections continued for 5 days. The third group was given penicillin according to the scheme described above with the addition of the intravenous phage treatment (5 ml/kg) every day for 5 days. The first phage injection was given 24 h after the infection. The 20 N. Chanishvili and Z. Alavidze animals were observed for 14 days, after which they were euthanized. Blood analysis was performed on days 1, 3, 5, 7, and 10 (Anikina 1982). The control animals demonstrated high temperature, weight loss, and pyuria. Two animals died after 4–5 days. Examination of the surviving animals demonstrated the development of multiple widespread abscesses including on the kidneys. S. aureus was isolated from all organ samples and urine. The 2nd group of rabbits treated with penicillin demonstrated the same symptoms as the control animals, two died on the 9th and 12th days; 5 days after infection all animals from this group started to produce pus in urine. Examination of the animal organs did not show any pathological changes. Bacteriological analysis of the organ samples, however, showed the presence of S. aureus. In the third group of rabbits undergoing combined treatment with penicillin and bacteriophage, all animals survived. A rise in temperature was observed as late as 3 days. After 7 days the blood samples appeared to be sterile. Dissection of the animals did not show any development of abscesses, and the presence of S. aureus was not seen by bacteriological analysis (Anikina 1982). Following animal trials, the phage was tested on 20 human volunteers with various types of acute and chronic staphylococcal infections. No side effects were observed. As a result of this, special permission was granted on April 11, 1979, allowing clinical trials on 250 patients per hospital, simultaneously in several hospitals. Altogether 653 patients were involved in the clinical trials, 355 men and 298 women, with 345 patients in the experimental group and 308 in the control group. 130 patients in the experimental group were treated with the intravenous phage preparation only; the other 215 received a combined treatment with the phage and antibacterial preparations commonly used in the medical practice (antibiotics, etc.). Patients in the control group were only treated with antibiotics. Usually intravenous staphylococcal phage (IVSP) was applied to patients having contraindications against antibiotics such as allergies, pregnancy, multiply resistant forms of staphylococcal infections, etc. The IVSP treatment was often applied after unsuccessful antibiotic therapy. The IVSP was mainly used intravenously (Meladze et al. 1981; Bochorishvili 1984; Chkhetia 1984; Samsygina and Boni 1984; Samsygina 1985). Only in cases of traumatic osteomyelitis was the phage applied intra-arterially (Tavberidze 1993). For intravenous use the IVSP was used in a dose of 0.5–1 ml per 1 kg of weight as transfusions combined with blood replacing compounds (saline solution, etc.). Higher doses (2 ml per 1 kg of weight) were applied rarely, e.g., in the cases of osteomyelitis. During the intravenous phage administration of the IVSP, the doctors did not observe any life-threatening side effects (Meladze et al. 1981; Chkhetia 1984). The results of the clinical studies were published by a group of doctors from the Institute of the Clinical and Experimental Surgery (Tbilisi, Georgia) – one of the organizations implementing clinical studies (Chkhetia 1984; Meladze et al. 1981). They performed a thorough analysis of the immune changes occurring during phage therapy. In total, 340 patients (253 males and 87 females) with unspecific festering diseases of the pleura and lungs were under observation. An experimental group received a complex treatment with the IVSP and antibiotics. A control group was treated with antibiotics only. Stable remission in the experimental group was attained on average in 53.5% of cases in contrast with the control group at Early Therapeutic and Prophylactic Uses of Bacteriophages 21

22.0%. In the experimental group, sequelae and lethal outcomes were observed in 2% of cases, while in the control, this figure was 4%. The authors indicated that 43 patients received intravenous phage transfusions together with antibiotics and topical treatment with bacteriophages. The dose for intravenous application was 0.5– 1.0 ml per kg of weight. No side effects were observed in the cases of intravenous phage transfusions; only in the cases of use of phages without antibiotics was a mild rise of temperature of 0.3–0.6 C observed. Local irritation of the bronchi epithelium was not noted. The duration of phage therapy was determined according to the X-ray analysis and clinical observations (Meladze et al. 1981; Chkhetia 1984). Interestingly, the microbiological analysis of the lung flora demonstrated dynamic changes in antibiotic susceptibility patterns. At the start of treatment, 64.4% of strains isolated from the experimental group showed resistance to antibiotics, compared to 66.7% in the control group. After implementation of phage therapy, drug resistance in the experimental group decreased to 60%, while in the control group, it increased to 73.8%. Resistance to staphylococcal bacteriophage was determined in 13.4% of cases (Chkhetia 1984). Dr. Nugzar Chkhetia (1984) described the results of treating 152 patients recov- ering from lung operations. A control group of 107 patients was treated with antibiotics alone, and 45 patients received antibiotics together with phages. Remis- sion and stabilization of the suppuration process in the experimental group was observed in 93% of cases, while in the control group, it was 80%. The frequency of post operational reinfection of the pleural cavity was 23% in the experimental group, compared to 67% in the control group. No lethal outcomes were observed in the experimental group, while a lethal outcome was observed in 8.4% of the group treated with antibiotics alone (Meladze et al. 1981; Chkhetia 1984). Phages were administered through various routes including local administration (tampons, bath- ing of cavities), inhalation, oral and parenteral, including intramuscular and intravenous injections. Phage preparations were applied as either liquid preparations or aerosols, with doses varying between 10 ml and 150 ml. The author recommended performing post-operational treatment by intra-pleural administration of phages. In this case the phage could be administered either via a drainage tube or by puncture. The dose of the liquid phage to be used could be determined by the remaining volume of pleural cavity, which usually varies between 10 and 300 ml. Prior to phage administration, the pleural cavity should be released of exudates by aspiration and then washed with a sterile saline solution mixed with a painkiller. Topical application of bacteriophage therapy did not cause any side effects. The duration of the topical phage therapy depended on the speed of the healing process. According to the author, however, it should not exceed 20 days (Chkhetia 1984). The IVSP was successfully applied to treatment and prophylaxis of staphylococcal post-traumatic infections of the long bones. The study refers to 45 cases in the experimental group, 6 of which were treated with the IVSP alone and 39 with IVSP in combination with antibiotics, and 50 in the control treated with antibiotics alone (Tavberidze 1993). After the clinical trials, Dr. Levan Tavberidze (1993) from the Institute of Traumatology (Tbilisi, Georgia) continued with the application of IVSP. Altogether 125 patients were treated, 109 of whom had been diagnosed with post- 22 N. Chanishvili and Z. Alavidze traumatic osteomyelitis, and of these, 62 had unconsolidated fractures. 69 patients received phage therapy (topically or intra-arterially) combined with antibiotics; 59 were treated with antibiotics alone. Prior to phage therapy, all patients had undergone unsuccessful generalized antibiotic and topical disinfecting therapies (Tavberidze 1993). The presence of S. aureus was proved by bacteriological analysis in 76% of cases. 13.1% cases were mixed infections with Proteus sp. In the majority of cases, the isolated strains showed resistance to multiple antibiotics. At the same time, these strains were susceptible to the IVSP (90.4%). The phage treatment was performed topically by introduction of the preparation into the fistula, festering cavities, soft tissues, etc. Prior to application of the phage preparation, the infected site was washed with 2% sodium bicarbonate solution. 69 patients received phage therapy prior to operation. Dynamic changes in the wounds were studied regularly by bacteriological analysis (105 tests). In 90% of cases, the wounds were cleaned of infection, and the festering and inflammation was reduced in comparison with the initial indexes. After the operation, phage therapy continued, but it was combined with antibiotic treatment. A mixture containing 1 g of antibiotic diluted in 20 ml of phage was administered into the wound by irrigation or by injection into the soft tissues localized around the wound. Altogether 155 patients received phage therapy in the post-operational period, among them 124 cases with osteomyelitis of the ankle where the phage administration was performed intra-arterially. Other patients were treated topically. Intra-arterial phage therapy was combined with antibiotics. Phage preparation was administered directly into the hip arteries. The effect of intra-arterial administration was understood as being more efficient than other methods. The clinical effect was obvious after 4–5 injections and was shown as a decrease of pain, normalization of temperature, reduction of inflammation, cleaning of the wound, improvement of the blood test parameters, etc. This effect was explained by concentration of high doses of the preparations in the soft tissues, which could not be achieved by other methods. Side effects were observed in two cases as a rise in temperature up to 39 C, which was easily released with painkillers. The most positive outcome was shown in the group receiving combined phage and antibiotic therapies (Bochorishvili 1984). Along with the improvement of clinical symptoms, a normalization of blood analysis, such as SOE (sedimentation of erythrocytes), and immune indices (phago- cytic activity of leukocytes) occurred due to the phage therapy (Samsygina and Boni 1984; Samsygina 1985). Samsygina (1985) concluded that the immunomodulating properties of the IVSP should have been considered as one of the most important properties of this preparation. Interestingly, after application of IVSP, the symptoms of the bacteremia in the majority of cases disappeared within 5 days. Among 149 children the bacteremia was observed in 53 cases. After the phage therapy, bacteremia remained in eight cases and after 7–10 days in only three cases. Mean- while, in the control group of patients, the bacteremia remained in 50% of cases even after 10 days of antibiotic therapy. Thus, the doctors concluded that the optimal duration of phage therapy in children was 7–10 days (Samsygina and Boni 1984; Samsygina 1985). Early Therapeutic and Prophylactic Uses of Bacteriophages 23

Forty-four children with mild symptoms of located festering inflaming disease (LFID) received intravenous phage treatment combined with topical therapy, without antibiotics. Ten patients were suffering with dacryocystitis, 12 with conjuncti- vitis combined with omphalitis, and 22 with omphalitis in combination with pyodermatitis. The IVSP was transfused every day for 4–5 days, and a positive outcome was observed in all 44 cases, with complete cure seen on days 5 through 77 depending on severity of infection and underlying diseases. The effect was observed earlier than in the control group (how much earlier is not reported). No relapses were seen. Infection was cured in the experimental group with mild LFID in 6.3 days and in the control group in 8.2 days. Intravenous phage therapy applied to the severe generalized forms of septicemia leads to a complete cure after around 17 days compared with 24 days in the control groups. At the same time, the doctors underlined that the entire duration of the hospital stay in both groups was usually about 40 days. This was explained by other medical factors affecting newborns (e.g., neurology problems, etc.). It is important to note that out of a total of 257 treated cases, 9 lethal outcomes were observed: 1 out of 149 in the experimental group (0.7%) and 8 out of 98 in the control group (8.2%) (Samsygina and Boni 1984; Samsygina 1985). No significant side effects of phage therapy were observed. After application of IVSP for treatment in the age group of 1 month to 1 year (38 children), only 2 (3%) reacted with an increase in temperature up to 38–39 C. This reaction was observed during transfusion and was alleviated by painkillers. These patients demonstrated a strong reaction to skin testing with staphylococcal anatoxin. The authors presumed that the reaction to IVSP was caused by sensitizing with derivatives of the bacterium, Staphylococcus. They suggested performing the skin tests with staphylococcal anatoxin and/or IVSP prior to the intravenous application of the bacteriophage preparation when treating young children (Samsygina 1985). In summary, the authors concluded that the IVSP preparation could be used as a supplement to antibiotic therapy for the treatment of staphylococcal infections of adults and children. The IVSP was recommended for use on patients with contraindications to antibiotic therapy. The IVSP lessened observations of toxic shock, shortened the healing period, improved sanitation of the initial infectious sites, and decreased the number of fatalities (Samsygina 1985). Following these studies, the staphylococcal phage was produced at the industrial production plant which was part of the Eliava Bacteriophage Institute. It was successfully provided, for intravenous use (infusions, transfusions, injections), in many clinics throughout the former Soviet Union for 20 years, until the collapse of the Soviet Union in 1990. Staphylococcal phage was mostly used for treatment of chronic septicemia, for treatment and prophylaxis of eye, ear, throat, and lung diseases, for healing burns, to treatment of the bacterial consequence of surgical operations on the bones and skull and for female infertility problems related to bacterial inflammation, etc. 24 N. Chanishvili and Z. Alavidze

Conclusions

According to Larry Goodridge (2013), who reviewed old Western literature dedicated to use of phage therapy, those studies revealed much diversity in the drawn-out results and conclusions. Goodridge (2013) underlines that comparisons of these studies are impossible due to lack of information regarding concentration of phage, numbers of different phages employed, method of preparation, diversity of administration routes, and the fact that in many of the reports no controls were included. A similar conclusion was made by Pavlova et al. (1973) who referred to prophylactic use of phages carried out in the Soviet Union in sites of high infection. In their review article, Pavlova et al. (1973) summarized the results of phage prophylaxis experiments carried out from 1934 to 1971 in 100 publications, 93 of which were published by Soviet researchers. The authors found that the results obtained by different researchers varied greatly. Thus, some researchers refer to different levels of decrease of disease in experimental groups varying from 1.2 times (Belikova 1941) to 46 times (Fisher 1949) when compared with the control group. Pavlova al (1973) explained such diversity of results as being due to methodological errors in the organization of the prophylactic measures. One of the major principles of such experiments was to provide quantitative and qualitative equity (i.e., similarity of epidemic conditions) in the establishing of experimental and control groups. This could be achieved by randomly selecting the people for the control group, carrying out of the same measures as in the experimental group but using a placebo, or by obligatory coding of the preparations used both in the experimental and control groups (Pavlova et al. 1973). The use of placebos and the coding of preparations was absent from most of the studies described in the previous sections. In addition, in some publications, such as Florova and Cherkass (1965), no control groups were used, and the efficiency of phage prophylaxis was estimated on the basis of com- paring the results of prophylactic phage treatment with figures from outbreaks obtained in previous years, when no prophylactic measure was carried out (e.g., Kagan et al. 1964). This approach was inappropriate primarily due to the epidemic cycles of this disease. According to Pavlova et al. (1973), in some publications, the numbers in the experimental and control groups are missing, which makes the results obtained insufficient for proper statistical analysis. In other articles (e.g., Belikova 1941; Antadze 1957), the numbers of children receiving bacteriophage preparation and those that formed the control group differed significantly. The same erroneous approach was made by many other authors (Pavlova et al. 1973). In this review article, the authors explained the lack of efficiency of the dysenteric phage preparations as being due to bad organization of prophylactic measures. One of the main reasons for the skeptical attitude to phage prophylaxis was the lack of reliable data on the phage preparation, which was used at the time the epidemics arose. The same errors were found also in designing and implementation of therapeutic experiments. No randomized, double-blind, placebo-supported experiments have been done before. This may be explained by the fact that the mass applications Early Therapeutic and Prophylactic Uses of Bacteriophages 25 were associated with war times when any treatment was considered as valuable and applicable. However, from the large amount of publications dedicated to use of phages for disease prevention, it is possible to conclude that, if the prophylactic measures are organized properly, the outcome could well be positive. For this purpose, consolidation of the efforts of the international scientific and medical communities is absolutely essential especially in the light of the background of growing antibiotic resistance.

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