BACTERIOPHAGE 2016, VOL. 6, NO. 4, e1251380 (8 pages) http://dx.doi.org/10.1080/21597081.2016.1251380
ADDENDUM
Fecal microbiota transplantation to fight Clostridium difficile infections and other intestinal diseases Karin Moellinga,b and Felix Broeckera,b,c a Max Planck Institute for Molecular Genetics, Berlin, Germany; bInstitute for Medical Microbiology, Unversity of Z€ urich, Z€ urich, Switzerland; cMax Planck Institute of Colloids and Interfaces, Potsdam, Germany; dDepartment of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
ABSTRACT
ARTICLE HISTORY
We have analyzed fecal bacterial and viral communities of a patient with recurrent C. difficile infection (rCDI) who was cured by fecal microbiota transplantation (FMT). The “Z€ urich Patient” experienced immediate cure and has remained free of symptoms for now over 5 y. Donor-similar bacterial compositions after 4.5 y post-FMT demonstrated sustainable engraftment of donor microbiota predominated by Bacteroidetes and Firmicutes bacteria. Appearance of beneficial species Faecalibacterium prausnitzii and Akkermansia municiphila was detected while disease-related Proteobacteria decreased. Stabilization of the microbiota took longer than expected from the rapidly improving clinical symptoms, suggesting the need for longer-lasting patient observation. The virome was mainly composed of Caudovirales bacteriophages but surprisingly also contained sequences related to a Chlorella giant virus that normally infects green algae not known to inhabitate the human intestine. FMT is highly effective against rCDI and is presently broadening its application to other conditions including inflammatory bowel disease (IBD). Here, we discuss the prospects and challenges of FMT against rCDI and other indications including a focus on bacteriophages.
Received 18 July 2016 Revised 13 October 2016 Accepted 18 October 2016
Introduction The incidence of potentially life-threatening rCDI increases worldwide.1 An estimated 453,000 infections and 29,300 related deaths occurred in the US in 2011.2 About 30% of patients experience recurrent infections following antibiotic treatment, often leading to chronic alternating patterns of treatment and relapse.1 Increasing unresponsiveness of rCDI to antibiotics fueled investigations on alternative treatment options, FMT being the most promising one.1,3 FMT replenishes the patients’ dysbiotic gut microbiota with the diverse microbes of healthy donor feces. Typically, fresh fecal suspensions are applied via nasal tubes, colonoscopy or enema.3 Already 1,700 years ago in ancient China a form of oral FMT, “yellow soup” prepared from fresh or fermented feces, was applied against food poisoning and diarrhea.4 Following the first report in modern scientific literature in 1958 by Eiseman et al., over 300 published cases of rCDI patients receiving FMT have
KEYWORDS
Chlorella giant virus; clinical trial; Clostridium difficile; fecal microbiota transplantation; microbiota; phage therapy; virome
shown cure rates of about 90% in the absence of severe adverse effects.3,5 In January of 2013, the first randomized, controlled clinical trial reported the superiority of FMT (via nasoduodenal tubes) to vancomycin.6 FMT cured 94% of rCDI patients, 81% after one treatment, compared to 23 or 31% of those receiving vancomycin with or without conjunctive bowel lavage, respectively. In April of 2013, the US Food and Drug Administration (FDA) classified human feces as Investigational New Drug (IND), which restricted the implementation of FMT to those physicians having an approved IND application.7 Opposition from gastroenterologists and patients caused the FDA on June 17th, 2013, to allow physicians to continue FMT against rCDI without IND but with informed consent from patient and donor.7 European authorities have followed this decision and now recommend FMT for antibiotic-refractory rCDI cases after more than 2 recurrent episodes.8 Donors without HIV or hepatitis
CONTACT Felix Broecker [email protected] Department of Microbiology, Icahn School of Medicine at Mount Sinai, 1 Gustave L. Levy Pl, New York, NY 10029, USA Addendum to: Broecker F, Klumpp J, Moelling K. Long-term microbiota and virome in a Z€urich patient after fecal transplantation against Clostridium difficile infection. Ann N Y Acad Sci 2016. http://dx.doi.org/10.1111/nyas.13100. © 2016 Taylor & Francis
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virus infections and with stool tested negative for C. difficile, Salmonella, Shigella, Escherichia coli, Yersinia enterocolitica and Campylobacter are eligible.9 Despite the long-known clinical efficacy of FMT, the precise mechanisms of action remain elusive. In 1958, Eiseman et al. suggested fecal inoculation to “reestablish the varied bacterial habitants of the bowel.”5 This assertion can now be studied in detail using advanced sequencing techniques that allow for quantitatively deciphering entire microbial communities, the microbiota.10 The healthy human intestine was shown to harbor 1014 cellular organisms of over 1,000 different species, mainly bacteria that conventionally are considered the most relevant component, mainly because other microbes are less studied. Yet, archaea, viruses, and fungi may also influence the functioning of the gut ecosystem.1,11 Phages are about 10 times more abundant than bacteria and comprise about 1,200 genotypes per individual.12,13 We and others have recently studied the fecal microbiota of rCDI patients post-FMT by deep sequencing to enhance our understanding of the gut microbes’ role – including viruses – in pathogenesis and cure of this disease.11
€ rich Patient The Zu We recently reported on the “Z€ urich Patient,” who after several antibiotic-refractory rCDI episodes experienced clinical cure within 2 w following FMT.14 It was surprising to what extent the Medical Faculty of the University of Z€ urich, in 2010, was opposed to FMT for a seriously ill rCDI patient. At that time FMT was not a recommended treatment and the physicians had to be convinced, mainly by the patient herself. The then recent publication of the human gut microbiome contributed to the decision, as it supported that feces may be the only feasible source to reinstall complex, healthy microbiota in the dysbiotic patient.10 FMT, the first such procedure in Switzerland, was eventually performed with informed consent obtained from patient and donor.14 Using metagenomic and 16S rRNA gene sequencing, we followed changes of bacterial and viral communities, respectively, in feces of patient and donor until 4.5 y post-FMT (Fig. 1A).11,14 The patient’s bacterial communities were highly variable over time for up to
Figure 1. Microbiota changes of the Z€ urich Patient. (A) Bacterial compositions determined by 16S sequencing11 shown as pie charts with indicated color code. (B) Relative abundances of butyrate-producing bacteria shown as bars. (C) Virome compositions determined by metagenomic sequencing14 shown as pie charts with indicated color code.
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7 months, but were highly donor-similar after 4.5 y, indicating stable microbiota engraftment.11 The patient had intriguingly high levels of Verrucomicrobia species 6 to 7 months post-FMT (Fig. 1B). This phylum was exclusively assigned to Akkermansia muciniphila (Verrucomicrobiaceae family). Notably, A. muciniphila was undetectable in the donor sample used for FMT, suggesting its origin from the patient. Yet, low levels in the donor 4.5 y post-FMT indicated that at the time of FMT it may have just been below the detection limit. High levels of A. muciniphila in the patient during the dysbiotic but symptom-free state suggested a role in maintaining functional microbiota. Indeed, an expansion of Verrucomicrobia/ Akkermansia has been frequently observed following successful FMT.11 Presence of A. muciniphila has been associated with healthy microbiota and support of intestinal integrity due to its capacity to produce the anti-inflammatory metabolite butyrate.15 Aside from A. muciniphila, most butyrate-producing species belong to the Firmicutes phylum, especially the subordinate Lachnospiraceae and Ruminococcaceae families whose presence correlates with healthy microbiota.16 Interestingly, both Lachnospiraceae and Ruminococcaceae in the Z€ urich Patient 6 to 7 months post-FMT were more abundant when compared to the healthy microbiota of donor and patient after 4.5 y (Fig. 1B). Depletion of butyrogenic bacteria is generally observed in rCDI patients, and successful FMT promotes an increase of Lachnospiraceae and Ruminococcaceae.11,16 A prominent Ruminococcaceae member is Faecalibacterium prausnitzii, a hallmark species of healthy gut microbiota.17 In the Z€ urich Patient, F. prausnitzii was detected at low levels 6 to 7 month post-FMT and showed an increase after 4.5 y (Fig. 1B). The abundance remained lower than in the healthy donor, despite higher levels of the superordinate Ruminococcaceae family in the patient. F. prausnitzii produces butyrate and low levels have been associated with intestinal inflammation.17 The role of F. prausnitzii during rCDI remain elusive, but the Z€ urich Patient suggests that reduced levels may well correlate with this disease. Virome analysis revealed the presence of dsDNA phages belonging to the Caudovirales order that stratified into Myo-, Podo- and Siphoviridae families (Fig. 1C). Phage communities in the patient were highly variable up to 7 months post-FMT. This could
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indicate their co-evolution with bacterial hosts,11 but may also reflect continued replenishment of phages from outside, e.g., by food consumption. Sequences related to the Paramecium bursaria Chlorella Virus-1 (PBCV-1), a large dsDNA virus of the Phycodnaviridae family infecting green algae, might represent a larger community of giant viruses with yet unknown impact on human health and disease.11 Aside from Phycodnaviridae, other giant viruses belonging to the Mimiviridae and Marseilleviridae families have been identified in stool samples of healthy individuals and patients with diarrhea and IBD.11,12 Although to date gut-resident giant viruses have not been associated with any disease, the oropharyngeal presence of the phycodnavirus Acanthocystis turfacea Chlorella Virus-1 (ACTV-1) has recently been linked to decreased cognitive abilities in mice and humans, and a Mimiviridae-related virus has been isolated from a patient with pneumonia.11 These findings strengthen the interest in further characterizing the largely unexplored community of giant viruses (the “megavirome”) in the intestine and reveal their potential impact on human health and disease. Donor specifications
Some properties of gut bacteria may require consideration when selecting a donor for FMT. Relatives are preferable donors with clinical cure rates of rCDI being higher than for unrelated donors (93% vs. 84%).3 This is likely due to the fact that the microbiota of a relative is more patient-similar as a result of genetic and environmental factors.11 Partners or spouses may be asymptomatically colonized with the same C. difficile strain and therefore be less preferable. Further complicating selection of appropriate donors, many parameters, including age, nutrition and antibiotic treatment, influence the gut microbiota.18 All these factors may have an impact on the outcome of FMT. In particular, the microbiota of obese individuals is pathologically altered19 and indeed, a predisposition to obesity may be transmissible by FMT even before clinical manifestation in the donor.20 Microbiota changes following FMT to treat metabolic syndrome have recently been analyzed with respect to single-nucleotide bacterial variants.21 Donor and recipient bacteria co-existed in the patients’ intestines for at least 3 months, whereby colonization success was greater for related “con-specific” strains than
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for newly introduced species. Furthermore, different recipients showed varying clinical outcomes even with the same donor, suggesting individual donor-recipient compatibilities. Thus, FMT may require personalized analysis of both donor and recipient beforehand, once selection criteria are better understood. FMT against other diseases
The good experience with rCDI has spawned the evaluation of FMT against other indications associated with dysbiotic microbiota, including IBD, metabolic syndrome, obesity, autoimmunity and anorexia nervosa.1,19,21 Supporting evidence comes mainly from small animal studies. When introduced into sterile mice, fecal microbiota of obese or lean individuals transmitted the respective donor phenotype, indicating obesity to be strongly determined by microbiota composition.19 Moreover, fecal transfer can influence the propensity for either explorative or more anxious behavior and affect the brain chemistry of mice.22 It is noteworthy in this context that the mammalian intestine harbors the enteric nervous system with 500 million neurons, compared to the central nervous system with 86 billion neurons.18 Moreover, the intestine produces 50% of the body’s dopamine and 95% of serotonin, neurotransmitters that both influence behavior. To what extent autism and other neurological disorders are influenced by the gut microbiota, and whether they can be transmitted or cured by FMT, remains to be elucidated. Results from human studies evaluating FMT against conditions other than rCDI have been largely disappointing to date.1,21 IBD, for instance, is associated with reduced Bacteroidetes and Firmicutes and expanded Proteobacteria, providing a rationale for FMT to be applied.1 Yet, dysbiosis is substantially milder than observed for rCDI.1 The still relatively complex microbiota of IBD patients may prevent sustainable donor stool engraftment, which necessitates repeated applications of FMT and concurrent antiinflammatory therapy against chronic tissue damage, such as neutralizing tumor necrosis factor signaling.1 Stable cure from IBD or metabolic syndrome may require complete abrasion of preexisting microbiota prior to FMT, but this would predispose patients to acquiring more severe diseases including C. difficile infection.
Viral dysbiosis
With about 1015 viruses and 1,200 genotypes per individual, the gut virome is at least as complex as the bacterial microbiota.12,13 Yet, dysbiosis commonly refers to pathological changes of bacterial communities, despite accumulating evidence for viral dysbiosis in certain diseases.10 Thus, similar to bacteria whose presence correlates with healthy microbiota such as F. prausnitzii that is investigated as a probiotic against IBD,17 there may be beneficial phages useful to treat diseases with viral dysbiosis. An aberrant expansion of phages is characteristic of IBD12 as well as obesity11 in humans and mice, respectively. Liberated phages have been proposed to contribute to IBD pathogenesis through bacterial lysis and concomitant release of pathogen-associated molecular patterns (PAMPs) that trigger inflammatory signaling.12 Phage activation also occurs during C. difficile infection and is likely a general characteristic of the inflamed intestine.11 Conversely, mucus-associated phages may protect from invading bacteria.23 Thus, gut phages can be both detrimental or beneficial and further virome studies are required to reveal the roles of individual phage types in human health and disease. We encountered some technical challenges during virome analyses. The amount of fecal material available was insufficient to isolate virions by cesium chloride density centrifugation that requires up to 500 g input material.13 Absence of a conserved region in viral genomes impedes promiscuous amplification of a marker like the bacterial 16S rRNA gene. Hence, we directly subjected non-amplified isolated total dsDNA from fecal samples to metagenomic sequencing.14 Thereby, only »0.1% of all sequences were assigned to known viruses, mainly Caudovirales dsDNA phages. Microviridae ssDNA phages that are also highly abundant in human feces12 could not be detected since the used Illumina sequencing pipeline was specific for dsDNA.14 The low abundance of phage sequences in the dsDNA preparations may partially reflect successful recovery of the patient, since healthy microbiota harbor fewer phages than those of patients with intestinal inflammation.11,12 When intended for virome analyses, we recommend storing large amounts (»500 g) of feces directly at ¡80 C. In our experience, there is no need for the addition of glycerol, ethanol or any other additives. Before sequencing, bacteria can be depleted by passing stool supernatant obtained from
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centrifugation successively through 0.45 mm and 0.22 mm filters.12 When little sample is available, centrifugation units with 100 kilodalton cutoff may be used to concentrate virions. Furthermore, virus genomes can be non-specifically amplified by multiple displacement amplification using phi29 DNA polymerase with degenerate primers prior to sequencing, which will increase the quality and information content of viral metagenomes and allow for detecting ssDNA phages.12 A particular challenge with fecal viromes is their high degree of inter-individual variability, which will complicate the identification of phages generally correlating with health or disease.11,12 Phage therapy for intestinal infections – lessons learned from clinical trials
The use of phages against bacterial infections was originally envisaged by Felix d’Herelle who discovered phages in 1917 – almost 100 y ago.24 However, soon thereafter, in 1928, the discovery of the antibiotic penicillin put an end to his hopes to establish phage therapy in the Western world. Antibiotics are easier to handle, act against a broader range of bacteria and completely out-competed phages as anti-bacterial agents – to the disappointment of d’Herelle. He and his team already noticed that phage cocktails were often required and successfully used those to treat cholera epidemics in India. Together with Georgi Eliava he founded the Eliava Phage Institute in Tbilisi, Georgia, that exists until today. This institute performed the only studies to date fulfilling the criteria of a controlled phase 3 trial.25 Preventive oral application of phages against Shigella dysentery significantly decreased disease incidence in children. Harald Br€ ussow and coworkers of the Nestle Research Center in Lausanne, Switzerland, recently reported on a clinical trial performed in Bangladesh aiming to treat children with E. coli diarrhea.26 Orally applied T4-like coliphages did not cause severe adverse effects and localized to the intestine. Yet, there was neither detectable phage replication nor an improvement in diarrhea outcome, likely due to low abundance of E. coli in the intestine and insufficient phage coverage. The Eliava and Nestle trials demonstrated that oral phages reach the intestine and are well-tolerated. However, some challenges emerged. For one, the intestinal target bacterium may not be abundant
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enough to allow for phage replication, as observed in the Nestle trial. Higher and/or repeated phage doses may be required for successful treatment. The usually narrow tropism of phages necessitates knowledge of the infecting strain, and this requires reliable molecular diagnostic tools. Challenges with phage therapy against C. difficile
Can phage therapy be implemented against rCDI? Some hurdles have to be overcome first. Many of the naturally isolated C. difficile phages are prone to enter the lysogenic cycle and have very narrow, strain-specific tropisms.11 Moreover, some of these phages have been shown to spread antibiotic resistance and bacterial virulence genes. These issues may be solved by engineered phages that express potent endolysins and have broader host range (Fig. 2). Recently, Timothy Lu and coworkers have shown that the tropism of lytic phages can be altered by exchanging the phage tails.27 Endolysins may even be used without needing the phage as carrier. Martin Witzenrath and coworkers have shown that inhalation of an isolated phagederived endolysin can limit lung infections with Streptococcus pneumoniae in mice, findings that are currently translated into a clinical trial.28 Of note, an endolysin isolated from a C. difficile-specific phage has been shown to induce lysis of the bacterium in vitro and may therefore be of therapeutic interest.29 Yet, lytic phages may not reach all target bacteria as their replication is self-limiting, and endolysins alone may be insufficient to inactivate mucosal biofilms of C. difficile (personal communication by Rob Meijers, EMBL Hamburg, Germany). Since the dysbiotic microbiota of rCDI patients is also characterized by antibiotic-induced overall changes of bacterial communities, such as expanded Proteobacteria,11,16 targeting those with phages may further support clinical success. Summary and outlook
FMT has emerged as highly efficient treatment against rCDI and is now recommended as rescue therapy for antibiotic-refractory cases in the US and Europe.7,8 Potential risks like the transmission of obesity or other dysbiotic conditions may be reduced by using cocktails of defined microbes.11 This necessitates identifying key species of healthy microbiota, of which F. prausnitzii and A. muciniphila are examples.15,17
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Figure 2. Proposed phage-assisted therapy for rCDI. Left: Natural phages frequently have narrow host ranges11,26 that may be altered through genetic engineering.27 Alternatively, isolated endolysins may be used.28,29 Right: Bacteroidetes and Firmicutes predominate healthy gut microbiota.11 C. difficile infection and antibiotic treatment causes detrimental Proteobacteria to expand.11 Both C. difficile and Proteobacteria may be targeted by engineered phages and/or endolysins, allowing for the microbiota to return to the healthy state, further facilitated by transferring beneficial bacterial species such as F. prausnitzii.17
However, viruses, not bacteria, are the most abundant entities in human feces, and dysbiotic viromes are characteristic of intestinal inflammation during rCDI, IBD and obesity.11,12 Phages are transmitted alongside bacteria during FMT, which can be considered almost a form of phage therapy, they shape bacterial communities and thereby possibly the outcome of this treatment.10 We thus expect the emerging viromics research field to reveal phages with therapeutic capacity against dysbiotic diseases. Defined microbial cocktails for targeted microbiota manipulation may be most potent when both specific bacteria and phages are included. Future progress may also involve combining therapeutic phages with antibiotics. Three recently isolated coliphages promoted lysis of E. coli biofilms in vitro.30 In combination, they completely inhibited bacterial growth and prevented emergence of phage-resistant mutants that were observed when single phages were used. Furthermore, phage-mediated biofilm disruption increased the efficacy of the antibiotic ciprofloxacin. Thus, in some cases phages and antibiotics may be most efficacious clinically when co-administered.
Another future aspect is the use of phages for disinfection. Phages, already widely added to food produce for conservation, may be used to sterilize hospital settings and medical equipment.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References [1] Khoruts A, Sadowsky MJ. Understanding the mechanisms of faecal microbiota transplantation. Nat Rev Gastroenterol Hepatol 2016; 13(9):508-16; Jun 22 [Epub ahead of print]; PMID:27329806; http://dx.doi.org/ 10.1038/nrgastro.2016.98 [2] Lessa FC, Winston LG, McDonald LC, Emerging Infections Program C. difficile Surveillance Team. Burden of Clostridium difficile infection in the United States. N Engl J Med 2015; 372:2369-70; PMID:26061850; http:// dx.doi.org/10.1056/NEJMc1505190 [3] Gough E, Shaikh H, Manges AR. Systematic review of intestinal microbiota transplantation (fecal bacteriotherapy) for recurrent Clostridium difficile infection. Clin Infect Dis 2011; 53:994-1002; PMID:22002980; http://dx. doi.org/10.1093/cid/cir632
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[4] Zhang F, Luo W, Shi Y, Fan Z, Ji G. Should we standardize the 1,700-year-old fecal microbiota transplantation? Am J Gastroenterol 2012; 107:1755; PMID:23160295; http://dx.doi.org/10.1038/ajg.2012.251 [5] Eiseman B, Silen W, Bascom GS, Kauvar AJ. Fecal enema as an adjunct in the treatment of pseudomembranous enterocolitis. Surgery 1958; 44(5):854-9; PMID:13592638 [6] van Nood E, Vrieze A, Nieuwdorp M, Fuentes S, Zoetendal EG, de Vos WM, Visser CE, Kuijper EJ, Bartelsman JF, Tijssen JG, et al. Duodenal infusion of donor feces for recurrent Clostridium difficile. N Engl J Med 2013; 368:407-415; PMID:23323867; http://dx.doi.org/10.1056/ NEJMoa1205037 [7] Critical views in gastroenterology & hepatology: Fecal microbiota transplantation: Where is it leading? Gastroenterol Hepatol (N Y) 2014; 10:307-9; PMID:24987314 [8] Debast SB, Bauer MP, Kuijper EJ, European Society of Clinical Microbiology and Infectious Diseases. European society of clinical microbiology and infectious diseases: update of the treatment guidance document for Clostridium difficile infection. Clin Microbiol Infect 2014; 2(20 Suppl):1-26; PMID:24118601; http://dx.doi.org/10.1111/ 1469-0691.12418 [9] Rohlke F, Stollman N. Fecal microbiota transplantation in relapsing Clostridium difficile infection. Therap Adv Gastroenterol 2012; 5:403-20; PMID:23152734; http://dx. doi.org/10.1177/1756283X12453637 [10] Qin J, Li R, Raes J, Arumugam M, Burgdorf KS, Manichanh C, Nielsen T, Pons N, Levenez F, Yamada T, et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 2010; 464:59-65; PMID:20203603; http://dx.doi.org/10.1038/nature08821 [11] Broecker F, Klumpp J, Moelling K. Long-term microbiota and virome in a Z€ urich patient after fecal transplantation against Clostridium difficile infection. Ann N Y Acad Sci 2016; 1372:29-41; PMID:27286042; http://dx.doi.org/ 10.1111/nyas.13100 [12] Norman JM, Handley SA, Baldridge MT, Droit L, Liu CY, Keller BC, Kambal A, Monaco CL, Zhao G, Fleshner P, et al. Disease-specific alterations in the enteric virome in inflammatory bowel disease. Cell 2015; 160:447-60; PMID:25619688; http://dx.doi.org/ 10.1016/j.cell.2015.01.002 [13] Breitbart M, Hewson I, Felts B, Mahaffy JM, Nulton J, Salamon P, Rohwer F. Metagenomic analyses of an uncultured viral community from human feces. J Bacteriol 2003; 185:6220-3; PMID:14526037 [14] Broecker F, Kube M, Klumpp J, Schuppler M, Biedermann L, Hecht J, Hombach M, Keller PM, Rogler G, Moelling K. Analysis of the intestinal microbiome of a recovered Clostridium difficile patient after fecal transplantation. Digestion 2013; 88:243-51; PMID:24335204; http://dx.doi.org/10.1159/000355955 [15] Khamsi R. A gut feeling about immunity. Nat Med 2015; 21:674-6; PMID:26121192; http://dx.doi.org/10.1159/ 000355955
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[16] Antharam VC, Li E, Ishmael A, Sharma A, Mai V, Rand KH, Wang GP. Intestinal dysbiosis and depletion of butyrogenic bacteria in Clostridium difficile infection and nosocomial diarrhea. J Clin Microbiol 2013; 51:2884-92; PMID:23804381; http://dx.doi.org/10.1128/ JCM.00845-13 [17] Miquel S, Martın R, Rossi O, Berm udez-Humaran LG, Chatel JM, Sokol H, Thomas M, Wells JM, Langella P. Faecalibacterium prausnitzii and human intestinal health. Curr Opin Microbiol 2013; 16:255-61; PMID:23831042; http://dx.doi.org/ 10.1016/j.mib.2013.06.003 [18] Moelling K. Nutrition and the microbiome. Ann N Y Acad Sci 2016; 1372:3-8; PMID:27285591; http://dx.doi. org/10.1111/nyas.13039 [19] Ridaura VK, Faith JJ, Rey FE, Cheng J, Duncan AE, Kau AL, Griffin NW, Lombard V, Henrissat B, Bain JR, et al. Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science 2013; 341:1241214; PMID:24009397; http://dx.doi.org/ 10.1126/science.1241214 [20] Alang N, Kelly CR. Weight gain after fecal microbiota transplantation. Open Forum Infect Dis 2015; 2:ofv004; PMID:26034755; http://dx.doi.org/10.1093/ofid/ofv004 [21] Li SS, Zhu A, Benes V, Costea PI, Hercog R, Hildebrand F, Huerta-Cepas J, Nieuwdorp M, Saloj€arvi J, Voigt AY, et al. Durable coexistence of donor and recipient strains after fecal microbiota transplantation. Science 2016; 352:586-9; PMID:27126044; http://dx.doi.org/10.1126/ science.aad8852 [22] Collins SM, Kassam Z, Bercik P. The adoptive transfer of behavioral phenotype via the intestinal microbiota: experimental evidence and clinical implications. Curr Opin Microbiol 2013; 16:240-5; PMID:23845749; http:// dx.doi.org/10.1016/j.mib.2013.06.004 [23] Barr JJ, Auro R, Furlan M, Whiteson KL, Erb ML, Pogliano J, Stotland A, Wolkowicz R, Cutting AS, Doran KS, et al. Bacteriophage adhering to mucus provide a non-host-derived immunity. Proc Natl Acad Sci U S A 2013; 110:10771-6; PMID:23690590; http://dx.doi.org/ 10.1073/pnas.1305923110 [24] Peitzman SJ. Felix d’Herelle and bacteriophage therapy. Trans Stud Coll Physicians Phila 1969; 37:115-23; PMID:4900376 [25] Sulakvelidze A, Alavidze Z, Morris JG Jr. Bacteriophage therapy. Antimicrob Agents Chemother 2001; 45:649-59; PMID:11181338 [26] Sarker SA, Sultana S, Reuteler G, Moine D, Descombes P, Charton F, Bourdin G, McCallin S, Ngom-Bru C, Neville T, et al. Oral Phage Therapy of Acute Bacterial Diarrhea With Two Coliphage Preparations: A Randomized Trial in Children From Bangladesh. EBioMedicine 2016; 4:124-37; PMID:26981577; http://dx.doi.org/10.1016/j. ebiom.2015.12.023 [27] Ando H, Lemire S, Pires DP, Lu TK. Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing. Cell Syst 2015; 1:187-96; PMID:26973885
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[28] Doehn JM, Fischer K, Reppe K, Gutbier B, Tschernig T, Hocke AC, Fischetti VA, L€offler J, Suttorp N, Hippenstiel S, et al. Delivery of the endolysin Cpl-1 by inhalation rescues mice with fatal pneumococcal pneumonia. J Antimicrob Chemother 2013; 68:2111-7; PMID:23633685; http://dx.doi.org/10.1093/jac/dkt131 [29] Mayer MJ, Garefalaki V, Spoerl R, Narbad A, Meijers R. Structure-based modification of a Clostridium difficile-
targeting endolysin affects activity and host range. J Bacteriol 2011; 193:5477-86; PMID:21803993; http://dx.doi. org/10.1128/JB.00439-11 [30] Dalmasso M, Strain R, Neve H, Franz CM, Cousin FJ, Ross RP, Hill C. Three New Escherichia coli Phages from the Human Gut Show Promising Potential for Phage Therapy. PLoS One 2016; 11:e0156773; PMID:27280590; http://dx.doi.org/10.1371/journal.pone.0156773
ADDENDUM
Fecal microbiota transplantation to fight Clostridium difficile infections and other intestinal diseases Karin Moellinga,b and Felix Broeckera,b,c a Max Planck Institute for Molecular Genetics, Berlin, Germany; bInstitute for Medical Microbiology, Unversity of Z€ urich, Z€ urich, Switzerland; cMax Planck Institute of Colloids and Interfaces, Potsdam, Germany; dDepartment of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
ABSTRACT
ARTICLE HISTORY
We have analyzed fecal bacterial and viral communities of a patient with recurrent C. difficile infection (rCDI) who was cured by fecal microbiota transplantation (FMT). The “Z€ urich Patient” experienced immediate cure and has remained free of symptoms for now over 5 y. Donor-similar bacterial compositions after 4.5 y post-FMT demonstrated sustainable engraftment of donor microbiota predominated by Bacteroidetes and Firmicutes bacteria. Appearance of beneficial species Faecalibacterium prausnitzii and Akkermansia municiphila was detected while disease-related Proteobacteria decreased. Stabilization of the microbiota took longer than expected from the rapidly improving clinical symptoms, suggesting the need for longer-lasting patient observation. The virome was mainly composed of Caudovirales bacteriophages but surprisingly also contained sequences related to a Chlorella giant virus that normally infects green algae not known to inhabitate the human intestine. FMT is highly effective against rCDI and is presently broadening its application to other conditions including inflammatory bowel disease (IBD). Here, we discuss the prospects and challenges of FMT against rCDI and other indications including a focus on bacteriophages.
Received 18 July 2016 Revised 13 October 2016 Accepted 18 October 2016
Introduction The incidence of potentially life-threatening rCDI increases worldwide.1 An estimated 453,000 infections and 29,300 related deaths occurred in the US in 2011.2 About 30% of patients experience recurrent infections following antibiotic treatment, often leading to chronic alternating patterns of treatment and relapse.1 Increasing unresponsiveness of rCDI to antibiotics fueled investigations on alternative treatment options, FMT being the most promising one.1,3 FMT replenishes the patients’ dysbiotic gut microbiota with the diverse microbes of healthy donor feces. Typically, fresh fecal suspensions are applied via nasal tubes, colonoscopy or enema.3 Already 1,700 years ago in ancient China a form of oral FMT, “yellow soup” prepared from fresh or fermented feces, was applied against food poisoning and diarrhea.4 Following the first report in modern scientific literature in 1958 by Eiseman et al., over 300 published cases of rCDI patients receiving FMT have
KEYWORDS
Chlorella giant virus; clinical trial; Clostridium difficile; fecal microbiota transplantation; microbiota; phage therapy; virome
shown cure rates of about 90% in the absence of severe adverse effects.3,5 In January of 2013, the first randomized, controlled clinical trial reported the superiority of FMT (via nasoduodenal tubes) to vancomycin.6 FMT cured 94% of rCDI patients, 81% after one treatment, compared to 23 or 31% of those receiving vancomycin with or without conjunctive bowel lavage, respectively. In April of 2013, the US Food and Drug Administration (FDA) classified human feces as Investigational New Drug (IND), which restricted the implementation of FMT to those physicians having an approved IND application.7 Opposition from gastroenterologists and patients caused the FDA on June 17th, 2013, to allow physicians to continue FMT against rCDI without IND but with informed consent from patient and donor.7 European authorities have followed this decision and now recommend FMT for antibiotic-refractory rCDI cases after more than 2 recurrent episodes.8 Donors without HIV or hepatitis
CONTACT Felix Broecker [email protected] Department of Microbiology, Icahn School of Medicine at Mount Sinai, 1 Gustave L. Levy Pl, New York, NY 10029, USA Addendum to: Broecker F, Klumpp J, Moelling K. Long-term microbiota and virome in a Z€urich patient after fecal transplantation against Clostridium difficile infection. Ann N Y Acad Sci 2016. http://dx.doi.org/10.1111/nyas.13100. © 2016 Taylor & Francis
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virus infections and with stool tested negative for C. difficile, Salmonella, Shigella, Escherichia coli, Yersinia enterocolitica and Campylobacter are eligible.9 Despite the long-known clinical efficacy of FMT, the precise mechanisms of action remain elusive. In 1958, Eiseman et al. suggested fecal inoculation to “reestablish the varied bacterial habitants of the bowel.”5 This assertion can now be studied in detail using advanced sequencing techniques that allow for quantitatively deciphering entire microbial communities, the microbiota.10 The healthy human intestine was shown to harbor 1014 cellular organisms of over 1,000 different species, mainly bacteria that conventionally are considered the most relevant component, mainly because other microbes are less studied. Yet, archaea, viruses, and fungi may also influence the functioning of the gut ecosystem.1,11 Phages are about 10 times more abundant than bacteria and comprise about 1,200 genotypes per individual.12,13 We and others have recently studied the fecal microbiota of rCDI patients post-FMT by deep sequencing to enhance our understanding of the gut microbes’ role – including viruses – in pathogenesis and cure of this disease.11
€ rich Patient The Zu We recently reported on the “Z€ urich Patient,” who after several antibiotic-refractory rCDI episodes experienced clinical cure within 2 w following FMT.14 It was surprising to what extent the Medical Faculty of the University of Z€ urich, in 2010, was opposed to FMT for a seriously ill rCDI patient. At that time FMT was not a recommended treatment and the physicians had to be convinced, mainly by the patient herself. The then recent publication of the human gut microbiome contributed to the decision, as it supported that feces may be the only feasible source to reinstall complex, healthy microbiota in the dysbiotic patient.10 FMT, the first such procedure in Switzerland, was eventually performed with informed consent obtained from patient and donor.14 Using metagenomic and 16S rRNA gene sequencing, we followed changes of bacterial and viral communities, respectively, in feces of patient and donor until 4.5 y post-FMT (Fig. 1A).11,14 The patient’s bacterial communities were highly variable over time for up to
Figure 1. Microbiota changes of the Z€ urich Patient. (A) Bacterial compositions determined by 16S sequencing11 shown as pie charts with indicated color code. (B) Relative abundances of butyrate-producing bacteria shown as bars. (C) Virome compositions determined by metagenomic sequencing14 shown as pie charts with indicated color code.
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7 months, but were highly donor-similar after 4.5 y, indicating stable microbiota engraftment.11 The patient had intriguingly high levels of Verrucomicrobia species 6 to 7 months post-FMT (Fig. 1B). This phylum was exclusively assigned to Akkermansia muciniphila (Verrucomicrobiaceae family). Notably, A. muciniphila was undetectable in the donor sample used for FMT, suggesting its origin from the patient. Yet, low levels in the donor 4.5 y post-FMT indicated that at the time of FMT it may have just been below the detection limit. High levels of A. muciniphila in the patient during the dysbiotic but symptom-free state suggested a role in maintaining functional microbiota. Indeed, an expansion of Verrucomicrobia/ Akkermansia has been frequently observed following successful FMT.11 Presence of A. muciniphila has been associated with healthy microbiota and support of intestinal integrity due to its capacity to produce the anti-inflammatory metabolite butyrate.15 Aside from A. muciniphila, most butyrate-producing species belong to the Firmicutes phylum, especially the subordinate Lachnospiraceae and Ruminococcaceae families whose presence correlates with healthy microbiota.16 Interestingly, both Lachnospiraceae and Ruminococcaceae in the Z€ urich Patient 6 to 7 months post-FMT were more abundant when compared to the healthy microbiota of donor and patient after 4.5 y (Fig. 1B). Depletion of butyrogenic bacteria is generally observed in rCDI patients, and successful FMT promotes an increase of Lachnospiraceae and Ruminococcaceae.11,16 A prominent Ruminococcaceae member is Faecalibacterium prausnitzii, a hallmark species of healthy gut microbiota.17 In the Z€ urich Patient, F. prausnitzii was detected at low levels 6 to 7 month post-FMT and showed an increase after 4.5 y (Fig. 1B). The abundance remained lower than in the healthy donor, despite higher levels of the superordinate Ruminococcaceae family in the patient. F. prausnitzii produces butyrate and low levels have been associated with intestinal inflammation.17 The role of F. prausnitzii during rCDI remain elusive, but the Z€ urich Patient suggests that reduced levels may well correlate with this disease. Virome analysis revealed the presence of dsDNA phages belonging to the Caudovirales order that stratified into Myo-, Podo- and Siphoviridae families (Fig. 1C). Phage communities in the patient were highly variable up to 7 months post-FMT. This could
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indicate their co-evolution with bacterial hosts,11 but may also reflect continued replenishment of phages from outside, e.g., by food consumption. Sequences related to the Paramecium bursaria Chlorella Virus-1 (PBCV-1), a large dsDNA virus of the Phycodnaviridae family infecting green algae, might represent a larger community of giant viruses with yet unknown impact on human health and disease.11 Aside from Phycodnaviridae, other giant viruses belonging to the Mimiviridae and Marseilleviridae families have been identified in stool samples of healthy individuals and patients with diarrhea and IBD.11,12 Although to date gut-resident giant viruses have not been associated with any disease, the oropharyngeal presence of the phycodnavirus Acanthocystis turfacea Chlorella Virus-1 (ACTV-1) has recently been linked to decreased cognitive abilities in mice and humans, and a Mimiviridae-related virus has been isolated from a patient with pneumonia.11 These findings strengthen the interest in further characterizing the largely unexplored community of giant viruses (the “megavirome”) in the intestine and reveal their potential impact on human health and disease. Donor specifications
Some properties of gut bacteria may require consideration when selecting a donor for FMT. Relatives are preferable donors with clinical cure rates of rCDI being higher than for unrelated donors (93% vs. 84%).3 This is likely due to the fact that the microbiota of a relative is more patient-similar as a result of genetic and environmental factors.11 Partners or spouses may be asymptomatically colonized with the same C. difficile strain and therefore be less preferable. Further complicating selection of appropriate donors, many parameters, including age, nutrition and antibiotic treatment, influence the gut microbiota.18 All these factors may have an impact on the outcome of FMT. In particular, the microbiota of obese individuals is pathologically altered19 and indeed, a predisposition to obesity may be transmissible by FMT even before clinical manifestation in the donor.20 Microbiota changes following FMT to treat metabolic syndrome have recently been analyzed with respect to single-nucleotide bacterial variants.21 Donor and recipient bacteria co-existed in the patients’ intestines for at least 3 months, whereby colonization success was greater for related “con-specific” strains than
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for newly introduced species. Furthermore, different recipients showed varying clinical outcomes even with the same donor, suggesting individual donor-recipient compatibilities. Thus, FMT may require personalized analysis of both donor and recipient beforehand, once selection criteria are better understood. FMT against other diseases
The good experience with rCDI has spawned the evaluation of FMT against other indications associated with dysbiotic microbiota, including IBD, metabolic syndrome, obesity, autoimmunity and anorexia nervosa.1,19,21 Supporting evidence comes mainly from small animal studies. When introduced into sterile mice, fecal microbiota of obese or lean individuals transmitted the respective donor phenotype, indicating obesity to be strongly determined by microbiota composition.19 Moreover, fecal transfer can influence the propensity for either explorative or more anxious behavior and affect the brain chemistry of mice.22 It is noteworthy in this context that the mammalian intestine harbors the enteric nervous system with 500 million neurons, compared to the central nervous system with 86 billion neurons.18 Moreover, the intestine produces 50% of the body’s dopamine and 95% of serotonin, neurotransmitters that both influence behavior. To what extent autism and other neurological disorders are influenced by the gut microbiota, and whether they can be transmitted or cured by FMT, remains to be elucidated. Results from human studies evaluating FMT against conditions other than rCDI have been largely disappointing to date.1,21 IBD, for instance, is associated with reduced Bacteroidetes and Firmicutes and expanded Proteobacteria, providing a rationale for FMT to be applied.1 Yet, dysbiosis is substantially milder than observed for rCDI.1 The still relatively complex microbiota of IBD patients may prevent sustainable donor stool engraftment, which necessitates repeated applications of FMT and concurrent antiinflammatory therapy against chronic tissue damage, such as neutralizing tumor necrosis factor signaling.1 Stable cure from IBD or metabolic syndrome may require complete abrasion of preexisting microbiota prior to FMT, but this would predispose patients to acquiring more severe diseases including C. difficile infection.
Viral dysbiosis
With about 1015 viruses and 1,200 genotypes per individual, the gut virome is at least as complex as the bacterial microbiota.12,13 Yet, dysbiosis commonly refers to pathological changes of bacterial communities, despite accumulating evidence for viral dysbiosis in certain diseases.10 Thus, similar to bacteria whose presence correlates with healthy microbiota such as F. prausnitzii that is investigated as a probiotic against IBD,17 there may be beneficial phages useful to treat diseases with viral dysbiosis. An aberrant expansion of phages is characteristic of IBD12 as well as obesity11 in humans and mice, respectively. Liberated phages have been proposed to contribute to IBD pathogenesis through bacterial lysis and concomitant release of pathogen-associated molecular patterns (PAMPs) that trigger inflammatory signaling.12 Phage activation also occurs during C. difficile infection and is likely a general characteristic of the inflamed intestine.11 Conversely, mucus-associated phages may protect from invading bacteria.23 Thus, gut phages can be both detrimental or beneficial and further virome studies are required to reveal the roles of individual phage types in human health and disease. We encountered some technical challenges during virome analyses. The amount of fecal material available was insufficient to isolate virions by cesium chloride density centrifugation that requires up to 500 g input material.13 Absence of a conserved region in viral genomes impedes promiscuous amplification of a marker like the bacterial 16S rRNA gene. Hence, we directly subjected non-amplified isolated total dsDNA from fecal samples to metagenomic sequencing.14 Thereby, only »0.1% of all sequences were assigned to known viruses, mainly Caudovirales dsDNA phages. Microviridae ssDNA phages that are also highly abundant in human feces12 could not be detected since the used Illumina sequencing pipeline was specific for dsDNA.14 The low abundance of phage sequences in the dsDNA preparations may partially reflect successful recovery of the patient, since healthy microbiota harbor fewer phages than those of patients with intestinal inflammation.11,12 When intended for virome analyses, we recommend storing large amounts (»500 g) of feces directly at ¡80 C. In our experience, there is no need for the addition of glycerol, ethanol or any other additives. Before sequencing, bacteria can be depleted by passing stool supernatant obtained from
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centrifugation successively through 0.45 mm and 0.22 mm filters.12 When little sample is available, centrifugation units with 100 kilodalton cutoff may be used to concentrate virions. Furthermore, virus genomes can be non-specifically amplified by multiple displacement amplification using phi29 DNA polymerase with degenerate primers prior to sequencing, which will increase the quality and information content of viral metagenomes and allow for detecting ssDNA phages.12 A particular challenge with fecal viromes is their high degree of inter-individual variability, which will complicate the identification of phages generally correlating with health or disease.11,12 Phage therapy for intestinal infections – lessons learned from clinical trials
The use of phages against bacterial infections was originally envisaged by Felix d’Herelle who discovered phages in 1917 – almost 100 y ago.24 However, soon thereafter, in 1928, the discovery of the antibiotic penicillin put an end to his hopes to establish phage therapy in the Western world. Antibiotics are easier to handle, act against a broader range of bacteria and completely out-competed phages as anti-bacterial agents – to the disappointment of d’Herelle. He and his team already noticed that phage cocktails were often required and successfully used those to treat cholera epidemics in India. Together with Georgi Eliava he founded the Eliava Phage Institute in Tbilisi, Georgia, that exists until today. This institute performed the only studies to date fulfilling the criteria of a controlled phase 3 trial.25 Preventive oral application of phages against Shigella dysentery significantly decreased disease incidence in children. Harald Br€ ussow and coworkers of the Nestle Research Center in Lausanne, Switzerland, recently reported on a clinical trial performed in Bangladesh aiming to treat children with E. coli diarrhea.26 Orally applied T4-like coliphages did not cause severe adverse effects and localized to the intestine. Yet, there was neither detectable phage replication nor an improvement in diarrhea outcome, likely due to low abundance of E. coli in the intestine and insufficient phage coverage. The Eliava and Nestle trials demonstrated that oral phages reach the intestine and are well-tolerated. However, some challenges emerged. For one, the intestinal target bacterium may not be abundant
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enough to allow for phage replication, as observed in the Nestle trial. Higher and/or repeated phage doses may be required for successful treatment. The usually narrow tropism of phages necessitates knowledge of the infecting strain, and this requires reliable molecular diagnostic tools. Challenges with phage therapy against C. difficile
Can phage therapy be implemented against rCDI? Some hurdles have to be overcome first. Many of the naturally isolated C. difficile phages are prone to enter the lysogenic cycle and have very narrow, strain-specific tropisms.11 Moreover, some of these phages have been shown to spread antibiotic resistance and bacterial virulence genes. These issues may be solved by engineered phages that express potent endolysins and have broader host range (Fig. 2). Recently, Timothy Lu and coworkers have shown that the tropism of lytic phages can be altered by exchanging the phage tails.27 Endolysins may even be used without needing the phage as carrier. Martin Witzenrath and coworkers have shown that inhalation of an isolated phagederived endolysin can limit lung infections with Streptococcus pneumoniae in mice, findings that are currently translated into a clinical trial.28 Of note, an endolysin isolated from a C. difficile-specific phage has been shown to induce lysis of the bacterium in vitro and may therefore be of therapeutic interest.29 Yet, lytic phages may not reach all target bacteria as their replication is self-limiting, and endolysins alone may be insufficient to inactivate mucosal biofilms of C. difficile (personal communication by Rob Meijers, EMBL Hamburg, Germany). Since the dysbiotic microbiota of rCDI patients is also characterized by antibiotic-induced overall changes of bacterial communities, such as expanded Proteobacteria,11,16 targeting those with phages may further support clinical success. Summary and outlook
FMT has emerged as highly efficient treatment against rCDI and is now recommended as rescue therapy for antibiotic-refractory cases in the US and Europe.7,8 Potential risks like the transmission of obesity or other dysbiotic conditions may be reduced by using cocktails of defined microbes.11 This necessitates identifying key species of healthy microbiota, of which F. prausnitzii and A. muciniphila are examples.15,17
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Figure 2. Proposed phage-assisted therapy for rCDI. Left: Natural phages frequently have narrow host ranges11,26 that may be altered through genetic engineering.27 Alternatively, isolated endolysins may be used.28,29 Right: Bacteroidetes and Firmicutes predominate healthy gut microbiota.11 C. difficile infection and antibiotic treatment causes detrimental Proteobacteria to expand.11 Both C. difficile and Proteobacteria may be targeted by engineered phages and/or endolysins, allowing for the microbiota to return to the healthy state, further facilitated by transferring beneficial bacterial species such as F. prausnitzii.17
However, viruses, not bacteria, are the most abundant entities in human feces, and dysbiotic viromes are characteristic of intestinal inflammation during rCDI, IBD and obesity.11,12 Phages are transmitted alongside bacteria during FMT, which can be considered almost a form of phage therapy, they shape bacterial communities and thereby possibly the outcome of this treatment.10 We thus expect the emerging viromics research field to reveal phages with therapeutic capacity against dysbiotic diseases. Defined microbial cocktails for targeted microbiota manipulation may be most potent when both specific bacteria and phages are included. Future progress may also involve combining therapeutic phages with antibiotics. Three recently isolated coliphages promoted lysis of E. coli biofilms in vitro.30 In combination, they completely inhibited bacterial growth and prevented emergence of phage-resistant mutants that were observed when single phages were used. Furthermore, phage-mediated biofilm disruption increased the efficacy of the antibiotic ciprofloxacin. Thus, in some cases phages and antibiotics may be most efficacious clinically when co-administered.
Another future aspect is the use of phages for disinfection. Phages, already widely added to food produce for conservation, may be used to sterilize hospital settings and medical equipment.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
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