FEATURE FEATURE wrong during the “first window of “My enemy’s enemy is opportunity” for phage therapy, says Richard Carlton, president and a my friend” director of Exponential Biotherapies in Port Washington, NY, USA, an 11-year-old biotechnology company Using phages to fight bacteria that is developing phage therapies for multidrug resistant bacteria. These and other early errors, acteriophages, viruses that prey descriptions of bacteriophages came coupled with the anecdotal nature of Bupon bacteria, typically attack only from Frederick Twort, a microbiologist clinical research at that time and the a single bacterial strain. This specificity, at the Brown Veterinary Hospital in discovery of chemical antibiotics, led to together with the killing capacity of London, in 1915. But Félix d’Herelle the rejection of phage therapy by most “phages”, says phage researcher Martin of the Institut Pasteur in Paris coined western doctors by the end of the Loessner, makes them the “natural the term bacteriophage in 1917 and 1940s, says phage researcher Carl enemies” of bacteria. “We are now soon afterwards began treating patients Merril, chief of the Laboratory of endeavouring to make this enemy our who had bacterial dysentery with an Biochemical Genetics at the US friend”, says Loessner, a professor of oral phage preparation. From then National Institutes of Health. But in food microbiology at the Swiss Federal until the mid 1940s, hundreds of eastern Europe and the former Soviet Institute of Technology in Zurich, papers were published describing the Union, phage therapy continued to be turning phages into potentially use of bacteriophages for the treatment widely used. Thousands of people were important allies in our battle against of dysentery and other human treated with individual phages and bacteria. infections, and commercial companies phage cocktails, many of them made at Most types of bacteriophages—the even offered off-the-shelf phage the Eliava Institute in Tbilisi, Georgia term means “bacteria eaters”—use preparations. —then the major phage production facility in the world. Phage preparations were used widely against dysentery by the military, for example, or applied topically to infected wounds, such as diabetic ulcers. Phage therapy turns out to be particularly good in this latter situation, says Betty Kutter, a professor of microbiology at Evergreen State College, Olympia, WA, USA, “because phages infect the bacteria near the Rights were not granted to include this image in electronic surface, and then multiply and go deeper for as long as there are bacteria media. Please refer to the printed journal. to infect”. By contrast, if antibiotics are applied locally, their concentration rapidly drops off with the distance from the wound surface, and systemic antibiotics won’t work because of the poor circulation typical of this type of wound, says Kutter, who has studied phages since 1963. Mzia Kutateladze is one of the scientists currently working at the Science Photo Library Eliava Institute on the molecular A bacteriophage uses spidery tail fibres (orange) to secure itself to the surface of the characterisation of phages for the bacterium (blue) treatment of infections. The first step in any phage therapy is to identify the molecules on their tail fibres to But results were mixed, due in large causative agent and check out which recognise and attach to their target’s part to a poor understanding of phage phage it is sensitive to, explains surface. The attached phage injects its biology. Phage preparations were used Kutateladze. Once that is done, a genetic material into the bacterium, in against bacteria insensitive to that suitable phage cocktail, which contains some cases killing its prey at this stage. particular phage or even against multiple phage clones active against The phage genes then commandeer the diseases that were not caused by one or several bacterial strains, is bacterium’s synthetic machinery to bacteria. In some cases, to prevent usually given to the patient, often in make phage components, which are bacterial contamination, manufacturers combination with or to complement assembled into new phage particles added oxidising agents to phage antibiotic therapy. For wounds and that are released when phage-encoded preparations that inactivated the phage. skin ulcers, the Eliava scientists have enzymes lyse the bacterial cell wall. Enthusiasm for phage treatments soon developed a biodegradable wound The idea of using phages to fight flagged in the west. Everything that dressing into which phage cocktails can bacteria is not new. One of the first could have been done wrong was done be incorporated. 624 THE LANCET • Vol 363 • February 21, 2004 • www.thelancet.com For personal use. Only reproduce with permission from The Lancet. FEATURE The phage preparations developed says Fischetti, “and clinical trials in in Tbilisi have been studied late 2005 if these studies go well.” extensively, both preclinically and All the approaches described clinically, say Kutateladze and above use phage or phage proteins Kutter. However, little of this as antibacterial agents. But scientists information has ever been published Rights were not granted to at PhageTech (Montreal, Canada) and even when details are available, include this image in hope that bacteriophages will lead reports rarely meet the full electronic media. Please refer them to new classes of antibiotics. requirements of a clinical trial. Thus, The traditional approach of says Kutter, “many US scientists to the printed journal. screening large libraries of molecules believe that phage therapy has been against bacteria for compounds with proven not to work rather than just antibacterial activity has had limited not proven to work, and they have success in recent years, says rejected much that has been done in PhageTech Vice-President Jinzi Wu, eastern Europe”. “so we thought, let’s use the natural Now though, phage therapy is enemies of bacteria to help us to undergoing a worldwide renaissance, identify their weak spots for target- driven largely by the emergence of Science Photo Library based antibiotic development”. multidrug-resistant bacteria. Dozens of bacteriophages (blue) can be seen Phages and bacteria have co-existed “Although many people now believe attacking an Escherichia coli bacterium for billions of years so bacterial it works, we still have to have the proteins that are attacked by phage proof”, in the form of extensive double- Vincent Fischetti, co-chair of the proteins during infection must be blind trials, says Kutter. Getting a phage laboratory of bacterial pathogenesis and critical to bacterial survival and/or therapy through the regulatory hurdles immunology at Rockefeller University growth, says Wu. If a small molecule laid down by national regulatory bodies (New York, USA) is interested in the candidate could be developed to inhibit will not be easy or cheap but clinical use of phage lytic enzymes. He these bacterial targets, it might provide Exponential Biotherapies has taken up has isolated phage enzymes directed a much-needed new kind of antibiotic. the challenge and is developing a phage against several gram-positive human Wu and colleagues collected phages therapy for vancomycin-resistant pathogens, including streptococci, from around the world able to infect Enterococcus faecium (VRE). In 2002, enterococci, and Bacillus anthracis and, Staphylococcus aureus, Streptococcus Carlton, Merril, and co-workers because most of these enzymes act only pneumoniae, and Pseudomonas aeruginosa successfully treated a bacteraemia against the bacterial species from which and sequenced the phage genomes. caused in mice by a clinical isolate of the phage was originally isolated, unlike Then, polypeptides derived from VRE with a phage that kills 95% of predicted open reading frames (ORFs) E faecium strains. This phage was well “phages infect the bacteria near from these genomes were tested for their tolerated in a phase I clinical trial and the surface, and then multiply ability to inhibit bacterial growth by phase II trials should start this year. and go deeper for as long as expressing the ORFs within bugs. From Phage researchers are also looking there are bacteria to infect” S aureus phages alone, the researchers for ways to use individual phage identified 31 distinct families of proteins as antimicrobials, particularly polypeptides that inhibit bacterial in the area of food safety and antibiotics they leave normal growth when expressed in S aureus agriculture. Martin Loessner and his commensal organisms untouched. (Nat Biotech 2004; 22: 185–91). colleagues in Zurich have isolated a Fischetti believes their main use will be “We used these growth-inhibitory lytic enzyme from a phage infecting in decolonising the carrier states of phage ORFs as a bait to fish out their Listeria monocytogenes, a dangerous these pathogenic organisms. If one interactive partners in bacteria”, contaminant of some soft cheeses. could treat members of a hospital continues Wu, and then screened This enzyme, which lyses bacterial cell community with a nasal spray of the chemical libraries for small molecules walls, kills more than 99% of listeria enzyme directed against streptococci, that inhibited these bacterial targets in cells when sprayed on to cheese. he says, the chances of nosocomial biochemical and functional assays. Loessner has put the lysin-encoding infection by this pathogen would be Several of these small molecules inhibit gene into Lactococcus lactis, bacteria reduced.