10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 2 3 4 5 6 7 8 9 728 resistant a stalker ofhospitalwards, methicillin- instancein which thefirst thorities reported ing: LastJuly, forexample, U.S. healthau- resistance. Somecasesarepar itation istheacceleratingcrisis of claim phagesfromthatdustbin. one ofmorethantwo dozenracingtore- based inPort Washington, New York, thatis ponential BiotherapiesInc.(EBI),afirm tory,” says RichardCarlton,presidentofEx- “Phages were relegated tothedustbinofhis- the therapy formorethanhalfacentury. came widespread, Western physicians ignored W W py More surprisingly, phagethera- oftheworld.this remotecorner the Stalineraisalive andwell in unique brandofmedicinefrom the lostskin. abroad fortreatmenttoreplace men were stable enoughtogo within afew weeks thewoods- eliminated theinfection,and ly patches,recent- PhageBioDerm ria. The businesscard–sized thatinfectbacte- phages, viruses es impregnated withbacterio- with novel biodegradable patch- tors covered theopenwounds ria’s naturalpredators. The doc- W therapy virtually unknown inthe the Soviet Unioncametotherescue. around thecorner. Then akinderlegacy of deep wounds. Septicshockseemedjust staphylococcus bacteriafrominvading the topreventwith antibioticsbutfailed the capital, Tbilisi, where doctorspliedthem men severely. The victimswere to rushed intensely radioactive two andburned ofthe once usedtopower remotegenerators,were rible mistake: The canisters,Soviet relics terly coldnight. outtobeater- That turned their campsitetowarm themselves onabit- touch. The menluggedtheobjectsbackto pair ofcanistersthatwere, oddly, hottothe the mountainsofGeorgia stumbled upona acquired fullresistance tovancomycin, of- T BILISI

orld War II,when theuseofpenicillinbe- est: They unleashedthebacte- est. After abriefflingwithphagesbefore

licensed forsaleinGeorgia, might beabouttostageacomebackinthe Driving phagetherapy’s potentialrehabil- The episodeshows thata toa Georgian doctorsturned — atrohg hrp,poerdi tlneaRsi,isattracting renewed attentioninthe West pioneered inStalin-era as a Russia, therapy, Last December, threewoodsmen in Staphylococcus aureus Stalin’s Forgotten Cure potential weapon against drug-resistant bugsandhard-to-treat infections iual chill- ticularly , hadalso that released intothewound asthey degraded. Irradiated. 5OTBR20 O 9 CEC www.sciencemag.org SCIENCE VOL 298 25 OCTOBER 2002 Georgian doctors covered thisman’s radiation burnswithpatches not tomention ourdigestive tracts,is phages; any exposed ofourbodies, surface seawater orsewage teemswithmillions of College inOlympia, Washington. A drop of gist ElizabethKutter ofEvergreen State onEarth,”dant lifeforms notesphagebiolo- going forthem.“They arethemostabun- that asamedicine,bacteriophageshave alot (seesidebar, p.730). market first theirway pathogensfood-borne willfind to that phageconcoctionsforlivestock and tance. For thesereasons,many expect experts after FDA approval tocounterbacterialresis- toalterphagestrains if itwillbepermissible ministration willregulate phageproducts,or is itevident how theU.S. Food andDrug Ad- or similarproductsintheUnitedStates.Nor ploring apotentialmarket forPhageBioDerm company inBaltimore, , that’s ex- says Alexander Sulakvelidze ofIntralytix, a are certainly notgoingtoreplacechemicals,” off theirpedestalformost infections.“Phages viral breedsareunlikely toknockantibiotics other openwounds, concurthatthese experts diabeticulcers,and as atreatmentforburns, arole resistant bacteriaandcouldsoonfind inBangalore. start-up launched GangaGenInc.,aphage-therapy dent of AstraZeneca Indiawho 2years ago presi- (“Ram”) Ramachandran,aformer is rapidly closing,” Janakiraman asserts window fornew ofopportunity ten Despite the uncertainties, proponentssayDespite theuncertainties, Although phagesoffer hopeagainstdrug- used asalast-ditchtreatment.“The N EWS F OCUS f P it was notedbiologist Félixd’Herelleof the that somehow killedbacteriain solution. But Tw 1915, Britishbacteriologist Frederick W. slayers must bemicrobesthemselves. In ing independently, concluded thatthebacteria 2 decadeslaterthatapairofscientists,work- could killthecholerapathogen. Itwasn’t until sewage-ridden GangesandJummarivers thatwaterHankin reported straightfromthe came in1896,when British chemistE.H. whiff ofthemicroscopicpredators The first A checkered past thus increasingpotency. their hosts,gainingstrengthinnumbersand they aremetabolized, phagesreplicatein loseeffectivenessAnd whereas drugs as fewer sideeffects thanuseofantibiotics. practice suggeststhatthisresultsinfar Anecdotal evidence fromdecadesofSoviet bacterialstrains. that homeinonparticular population andleave therestintact.” can killoff asmallfractionofthemicrobial Kutter says:they “Phages specific, arevery Sulakvelidze. And unlike mostantibiotics, y car be awonderful way togo.” at Harvard University. “Phagetherapy could Schaak oftheRowland InstituteforScience amous: He and his wife coined the term bac- amous: Heandhis wifecoinedtheterm asteur InstituteinParis who madethecritters ou ort describedan“ultramicroscopicvirus” ort pet EBI, the fastest companyEBI, thefastest off theblocks, bombs Phages arelike minusculesmart an ed withthem.“Mothernaturegives endless sourceofphages,” says rity inthe Western world. approach slipsbackintoobscu- reach themarket—or whether the therapies againsthumandiseases mine how phage quickly thefirst cause itsexperiences coulddeter- are watching EBIclosely, be- Montreal, Canada.Heandothers in president ofBiophagePharma w really areliving inabrave new in themiddleofnext year. “We clinical trialinpatientswith VRE enterococci andplanstolauncha against vancomycin-resistant healthy volunteers ofaphage has completedsafetytestingin tive toantibiotics,” says Diane something, have somealterna- comeback. “We needtodo orld,” says Toney Ilenchuk, vice Many arerootingfor a

CREDIT: DAVID JIKIA N EWS F OCUS 65 teriophage in 1916 after d’Herelle isolated an by in the suspensions used to culti- tinued its pioneering work, including the de- 64 “anti-Shiga” microbe from the feces of pa- vate phages, and they were rarely tested be- velopment in the 1940s of phages against 63 tients with dysentery and grew it in the bac- fore use to see if the phages were viable. anaerobic infections such as gangrene. Soviet 62 terium that causes the disease. But whereas Western physicians abandoned authorities placed a high value on the Eliava 61 D’Herelle was also the first to compre- the fickle medicine, Soviet scientists kept Institute’s work. When it came to ordering 60 hend the promise that phages held as a dis- the faith. new equipment and supplies of enzymes, 59 ease treatment. In 1919, he and his col- says microbiologist Mzia Kutateladze, who 58 leagues made a phage preparation for a 12- Stalin’s antibiotic alternative joined the institute in the late 1980s, “we got 57 year-old boy with severe dysentery. After Sunlight streams through a picture window whatever we wanted.” 56 guzzling 100 times the intended dose to into an office suffused with the yeasty smell The Soviet military was perhaps the 55 check its safety—“the first clinical safety of agar as Amiran Meipariani removes a log- biggest consumer of phage preparations, 54 trial,” jokes Sulakvelidze—they gave the di- book from a desk drawer. The silver-haired many of which were produced in Russia— 53 luted preparation to the boy, who recovered and -moustached bacteriologist scrolls down and still are—according to Georgian tech- 52 fully within a few days. a record in cramped Cyrillic 51 Over the next several years, d’Herelle handwriting of the last batches 50 helped set up phage-therapy trials across the of medicinal bacteriophages 49 globe. “He would go to villages and observe shipped abroad by the Eliava 48 who was recovering on their own from an ill- Institute here in Tbilisi. On the 47 ness, isolate phages from these people, and wall behind him is a 1930s 46 grow them in the lab,” says Ramachandran. photograph of the man who 45 was off to a flying start, and it started it all in Georgia, a dash- 44 gained in popularity after the 1925 publication ing young scientist with oiled 43 of Arrowsmith, a novel by Sinclair Lewis in black hair and deep-set eyes. 42 which a doctor deploys phages against an out- Under Giorgi Eliava’s intense 41 break of bubonic plague in the West Indies. gaze, Meipariani, who has 40 Back then, “phages seemed like a miracle worked here for 45 years, puts 39 answer to many devastating infectious dis- a finger on the most recent en- 38 eases,” says Kutter. The drug giant Eli Lilly tries in his log: 88,600 phage 37 and a plethora of entrepreneurs piled into the tablets for intestinal illnesses 36 phage business, but their record was spotty. and 497,000 tablets for prophy- Heady days. Félix d’Herelle (seated) and Giorgi Eliava (right) 35 In some patients the concoctions worked laxis against , both in Tbilisi shortly before Eliava was shot. 34 well, whereas in many others they had no ef- shipped to Central Asia in 33 fect. The mixed results were grist for a 1989. That was the beginning of the end of niques. “Antibiotics were expensive, while 32 damning critique of phage therapy from the the Eliava Institute’s golden era. phage preparations were very cheap,” ex- 31 American Medical Association in 1934. The Soviet enterprise got under way in plains Meipariani. The military’s enthusiasm 30 Phage enthusiasts are quick to disassoci- 1923, when Eliava, who had spent 5 years did not ebb after the Soviet meltdown. Dur- 29 ate modern approaches from the field’s early with d’Herelle in Paris, founded a bacterio- ing the civil war in the early 1990s, Georgian 28 days. Little was known then about phages or logical research center with the blessing of soldiers fighting in the breakaway Abkhazia 27 bacteria, so patients often took phages that Soviet dictator Josef Stalin. Eliava’s phage region carried spray cans filled with phages 26 were not suited to their infections. In addi- program got a big boost in 1933, when against five bugs: Staphylococcus aureus, 25 tion, says EBI’s Carlton, “they didn’t purify d’Herelle left Yale University to join his pro- , Pseudomonas aeruginosa, 24 these products well enough.” Preparations tégé in Tbilisi. He stayed until tragedy Streptococcus pyogenes, and Proteus 23 were often loaded with endotoxins produced struck a few years later: Eliava fell into dis- vulgaris. Phage preparations were also widely 22 favor with the dreaded available in many Russian cities alongside 21 Lavrenty Beria, later antibiotics. And in a handful of towns such 20 head of the KGB, and as Tolyatti, an auto-manufacturing center, 19 was executed. The dev- clinics “rarely used antibiotics,” instead rely- 18 astated institute eventu- ing almost exclusively on phages, says Zem- 17 ally recovered and con- phira Alavidze, a microbiologist at the Eliava. 16 By the time the Soviet Union dis- 15 solved in 1991, the Eliava had only the 14 means to produce phages for a still- 13 thriving domestic market in newly inde- 12 pendent Georgia. This minimal produc- 11 tion, says Meipariani, has helped “pre- 10 serve the tradition.” Cooking up phages 9 means more than following a recipe, he 8 says: “You need a good mind and good 7 hands.” Visitors concur. “There is really 6 no substitute for their collective experi- 5 ence over the past 70 years,” says Tony 4 Smithyman, managing director of SPS, a 3 Old Georgian recipe. Microbiologist Zemphira Alavidze examines phage-therapy company in Sydney, Aus- 2 bacterial colonies decimated by phages. At the Eliava Institute, tralia. He’s one of many Westerners who

1 CREDITS:A. OF COURTESY (TOP) SULAKVELIDZE; MUTSUMI STONE (BOTTOM) hardy strains are cultivated and refrigerated the old-fashioned way. have made a pilgrimage to Tbilisi to

www.sciencemag.org SCIENCE VOL 298 25 OCTOBER 2002 729 N EWS F OCUS 65 64 Food and Agriculture:Testing Grounds target the five or six serogroups most commonly associated with 63 human illness,” says Intralytix co-founder Alexander Sulakvelidze, who 62 For Phage Therapy directed the State Microbiology Laboratory in Tbilisi, Georgia, before 61 Last month, the U.S. Food and Drug Administration tightened another emigrating to the United States a decade ago. 60 screw in its effort to curb the spread of antibiotic resistance from the Fresh-cut produce might also be candidates for phage treatment: 59 burgeoning use of agricultural drugs. The agency aired draft regulations A team at the U.S. Department of Agriculture (USDA), working with 58 requiring manufacturers to test potential livestock pharmaceuticals for Intralytix, has found that phages are more effective than chlorine at 57 their ability to help pathogens acquire resistance to human drugs. But ridding cut fruits and vegetables of Salmonella.At least two dozen 56 farmers are concerned that they other phage firms worldwide are hoping to get a 55 could be left with fewer weapons to foothold in the food and livestock market. 54 combat Listeria and other food- Shadowing the corporate push is surging academic 53 borne pathogens that cause several interest in using phages to improve food safety. For ex- 52 hundred deaths each year in the ample, a team led by microbiologists Donna Duckworth 51 United States alone. “When farmers and Paul Gulig of the University of Florida, Gainesville, 50 are told they can’t use any anti- Image not has isolated phages against Vibrio vulnificus,a bacterium 49 biotics used in humans, they say, sometimes found in raw oysters that can trigger severe 48 ‘What do we use?’” says Toney available for illness in people. The researchers have used these phages 47 Ilenchuk. His firm, Biophage Pharma online use. to cure infected mice and are trying to harness the 46 in Montreal, Canada, believes it has phages for depurating oysters 45 part of the answer: bacteriophages for human consumption. In a 44 against Salmonella and pathogenic similar vein, Elizabeth Kutter’s 43 strains of Escherichia coli. team at Evergreen State College 42 Ilenchuk and other advocates in Olympia, Washington, is 41 are also eyeing the use of phages— working with USDA to find 40 viruses that attack bacteria—in A man and his phage. Alexander phages that can clear the deadly 39 food processing. The Baltimore, Sulakvelidze shows off a phage E. coli O157:H7 from cattle 38 Maryland–based firm Intralytix preparation sold in Georgia; elec- guts. The strain doesn’t seem to 37 already has a permit from the U.S. tron micrograph of bacterio- bother cows, but outbreaks 36 Environmental Protection Agency to phages that kill Salmonella. traced to undercooked ham- 35 test a phage against the bacterium burgers and unpasteurized fruit juices have killed scores of people. 34 in a food-processing plant, although in this trial All these potential applications would require regulatory ap- 33 the phages cannot be applied to surfaces that come into contact with proval. But enthusiasts hope that the very ubiquity of phages will 32 food. The company hopes to have its anti-Listeria phage on the market make them an easy sell. They are “the ultimate clean, green, 31 by early next year. Intralytix is also developing phage preparations to ecofriendly disease-control system,” pitches Tony Smithyman of 30 spray on eggs to reduce Salmonella contamination. It would be too SPS, a phage-therapy firm in Sydney, Australia. “We’re working in 29 daunting to go after all 2400 or so Salmonella serogroups, “but we can tune with nature.” –R.S. 28 27 26 learn the art of phage therapy. in the early 1940s, molecular biologists ex- league Sankar Adhya wondered whether 25 The Eliava Institute was not alone in pur- ploited the simplicity and ease of handling some particularly hardy strains might evade 24 suing such therapies, as phage centers of a few lab phages to do the following: clearance. If so, these could be harvested 23 sprang up elsewhere in Eastern Europe. Per- confirm that DNA is the genetic material, and studied and perhaps serve as the basis 22 haps the most important data in the English show that messenger RNA is involved in its for an improved therapy. To find out, the re- 21 literature on the therapy’s effectiveness translation, reveal that the unit of recombi- searchers proposed injecting mice with bil- 20 come from the Institute of Immunology and nation is the nucleotide, and clarify how lions of lambda phages and seeing if any 19 Experimental Therapy in Wrocl–aw, Poland. genes are turned on and off. But the main- persisted hours later. Teaming up with EBI’s 18 Researchers there compiled a detailed report stream view on phage therapy was summa- Carlton, they found that phage mutants that 17 on the successful treatment of more than rized in a passage from Gunther Stent’s clas- were around many hours longer than run-of- 16 500 patients with bacterial infections in the sic 1963 textbook Molecular Biology of the-mill phages were much more effective at 15 mid-1980s, but the results appeared in the Bacterial Viruses: “Just why bacteriophages, rescuing mice from otherwise lethal infec- 14 obscure Archivum Immunologiae et Thera- so virulent in their antibacterial action in tions. Their 1996 report, in the Proceedings 13 piae Experimentalis and only recently were vitro, proved to be so impotent in vivo has of the National Academy of Sciences (Vol. 12 excavated for wider dissemination. The never been adequately explained.” 93, p. 3188), was widely hailed as a basis 11 Wrocl–aw institute is itself experiencing a re- Stent suggested that the therapy failed for selecting promising phage strains. For 10 naissance, busily culturing medicinal phages because antibodies mop up phages infused the first time in more than half a century, 9 and forging ties with Western labs. into the body. In the early 1970s, a young re- Western experts were taking the disparaged 8 searcher at the U.S. National Institutes of approach seriously again. 7 Promises and perils Health (NIH), Carl Merril, tested that notion In a sign of the changing times, the Cold 6 Whereas antibiotics relegated phage therapy on a germ-free colony of mice using a com- Spring Harbor Laboratory in New York, 5 to a historical footnote in the West, bacterio- mon lab phage, lambda. His group discov- once a powerhouse in basic research on bac- 4 phages themselves, particularly the lunar ered that before the mice could even develop teriophages, will hold its first-ever Banbury 3 lander–shaped T4 that infects E. coli, be- antibodies, the phages were cleared from the meeting on phage therapy next month. How- 2 came the darlings of biologists. Beginning bloodstream, primarily by the spleen. ever, there are still big gaps in our under-

1 with Max Delbrück’s famous “phage group” Several years later, Merril and NIH col- standing of how phages work: Exquisite CREDITS: SAM KITTNER (TOP)

730 25 OCTOBER 2002 VOL 298 SCIENCE www.sciencemag.org N EWS F OCUS 65 studies of phage genetics have revealed little Such a master phage could be subtly tin- versal view. Kutter, for one, argues that 64 about how these viruses behave in their natu- kered with, for example by altering a single Eastern European findings on the use of 63 ral environments or when introduced into the gene that affects host susceptibility. Modi- phages to treat conditions such as diabetic 62 human body. “Decades of neglect of phage fied strains presumably would take less time ulcers and osteomyelitis, in which poor cir- 61 biology have left us woefully unprepared to and money to approve, Adhya says. culation can render antibiotics toothless, 60 take rapid steps in such uses as therapeutics,” Some scientists hope to bypass these is- “are particularly impressive and incontro- 59 says Ry Young, a phage biologist at Texas sues altogether by extracting the active com- vertible. They have excellent cure rates.” 58 A&M University in College Station. ponents from phages. For example, Vincent But it seems that message isn’t reaching the 57 What is known is that phages come in two Fischetti and his team at Rockefeller Univer- right ears. “We just get a blank stare when 56 flavors. “Lytic” phages infect a bacterium, sity in New York City describe in the 22 Au- we talk to regulators,” says Ilenchuk, who 55 hijack its DNA, and replicate madly until gust issue of Nature how they used a phage’s says his firm, Biophage Pharma, will target 54 the bacterium’s cell wall gives out and it ex- lytic enzyme to kill the anthrax bacterium in “compartments” such as the mouth or in- 53 pires: the killing mechanism common for all the test tube. In a similar vein, Young’s lab at testines rather than dive into injectibles. 52 phages. Lytic phages are ideal for therapy. Texas A&M reported last year in Science (22 FDA has not yet issued written guidance 51 But half of all phages, it is thought, are June 2001, p. 2326) that one type of phage on how it intends to regulate phage therapy. 50 “temperate,” meaning that they often inte- makes peptides that act like penicillin, block- But many unknowns will be cleared up as 49 grate their DNA into that of their host. “You ing cell-wall synthesis in bacteria and caus- EBI takes its VRE phage through trials. 48 wouldn’t know the phage is there,” says ing the cell to explode when it tries to divide. “They are blazing the trail for us,” says Asher 47 Adhya, because they hibernate in the form of Some companies are trying to exploit Wilf, who last year founded Phage Biotech 46 genetic code before the viral DNA tears itself such eccentricities of phages in their quest Ltd. in Rehovot, Israel. Navigating through 45 free again and the virus begins replicating— for new drugs. For instance, PhageTech in the regulatory waters will be “a big chal- 44 sometimes taking some of the host DNA St. Laurent, Canada, is studying a myriad of lenge,” says Ilenchuk. “It would take just one 43 with it. At the same time, temperate phages phage “killer” proteins that derail the host of us to screw it up,” he says, recalling the 42 can protect their host from attack by other troubles encountered in the 41 phages. And they can abet pathogens: Cer- early days of bringing blood 40 tain temperate phages carry the genes for substitutes to the market. 39 the toxins released by bacteria that cause FDA’s emerging stance could 38 diphtheria and cholera, for example. also determine if and when 37 One serious concern is that a temperate large pharmaceutical compa- 36 phage could make off with host genes con- nies get into the game. “Big 35 nected with virulence or resistance. These pharma is waiting to see 34 phages could, in principle, wreak havoc by Image not proof of concept before they 33 integrating such genes into a new host. “I available for do deals,” says PhageTech 32 would worry about the transference of such co-founder Michael DuBow, 31 genes,” says Harvard’s Schaak. After all, she online use. now at the Université Paris- 30 asks, “Why would a phage wipe out its Sud XI in Orsay, France. 29 host?” Schaak speculates that some phages In addition to regulatory 28 that are lytic in the test tube could acquire and scientific uncertainties, 27 the genes to turn temperate in the body. Al- phage enthusiasts might face 26 though most experts discount that possibility, one more hurdle: public ac- 25 Sulakvelidze in informal conversations with Back to the future. More than a century after a British chemist ceptance. The time is ripe to 24 FDA officials understood that firms must caught the first whiff of phage in water from the Ganges River, start educating the public 23 guarantee that their phages are stably lytic. the firm GangaGen is hoping to put Bangalore on the phage- about phages, says Ilenchuk. 22 “We’re doing much more rigorous character- therapy map. “We have to get the message 21 izations of phages than has been done in the across that phages are every- 20 past,” he says, including DNA sequencing. metabolism to make it easier for the phages where.” He and others assiduously avoid the 19 Another hurdle that could make or break to reproduce. It is now screening libraries of “v” word. Rather than refer to them as virus- 18 phage therapy is that bacteria inevitably de- small molecules for killer protein analogs es, says Schaak, “I call phages a natural deliv- 17 velop resistance to phage strains just as they that could act as antibiotics. And Schaak is ery system.” Others prefer a straight-shooting 16 do to antibiotics. The problem might not be taking a novel tack. Earlier this year, she and approach. “Phages are viruses, and if they are 15 as severe for would-be phage therapists: A a few colleagues launched MicroStealth to be used as therapeutic agents, we need to 14 study from the early 1980s found that muta- Technologies, which aims to use phages as respect their origin and use our modern sci- 13 tions conferring resistance in E. coli oc- delivery vehicles for antimicrobial peptides entific methods to assure that they are safe,” 12 curred less frequently following phage ther- that are only active inside bacterial cells. says NIH’s Merril. 11 apy than they did following antibiotic thera- No matter how it’s sold, most experts be- 10 py. And proponents say it’s much easier to Phage futures? lieve that in light of the increasing perils of 9 tackle resistance with phages than with Although the experiences in Georgia and antibiotic resistance, the once-scorned Soviet 8 drugs. “You can generate a new phage vari- elsewhere in Eastern Europe have helped therapy will ultimately find a niche in mod- 7 ant in a week” by selecting those that don’t establish sound methods for selecting and ern Western medicine. A half-century of 6 lose virulence in culture, says Ramachan- cultivating phages, it’s unclear how much of antibiotics usage has taught us that “you 5 dran, who envisions a regulatory process in that data will be useful to companies in- cannot win the war against bacteria,” says 4 which panels of phages are put through clin- tending to bring phage therapy to the West. Sulakvelidze. But with phages, he says, “at 3 ical trials for FDA approval. Adhya’s team, “I’m not knocking the work in the East,” least you can try to shift the ecological bal- 2 meanwhile, is developing a “master” phage asserts EBI’s Carlton, “but the FDA pretty ance in our favor.”

1 CREDIT: GANGAGEN INC. that could undergo rigorous FDA review. much has to discount it.” That’s not a uni- –RICHARD STONE

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