Oral Phage Therapy of Acute Bacterial Diarrhea With Two Coliphage Preparations: A Randomized Trial in Children From Bangladesh
Shafiqul Alam Sarker, Shamima Sultana, Gloria Reuteler, Deborah Moine, Patrick Descombes, Florence Charton, Gilles Bourdin, Shawna McCallin, Catherine Ngom-Bru, Tara Neville, Mahmuda Akter, Sayeeda Huq, Firdausi Qadri, Kaisar Talukdar, Mohamed Kassam, Michèle Delley, Chloe Loiseau, Ying Deng, Sahar El Aidy, Bernard Berger, Harald Brüssow, Shafiqul Alam Sarker, Shamima Sultana, Gloria Reuteler, Deborah Moine, Patrick Descombes, Florence Charton, Gilles Bourdin, Shawna McCallin, Catherine Ngom-Bru, Tara Neville, Mahmuda Akter, Sayeeda Huq, Firdausi Qadri, Kaisar Talukdar, Mohamed Kassam, Michèle Delley, Chloe Loiseau, Ying Deng, Sahar El Aidy, Bernard Berger, Harald Brüssow
Abstract
Background: Antibiotic resistance is rising in important bacterial pathogens. Phage therapy (PT), the use of bacterial viruses infecting the pathogen in a species-specific way, is a potential alternative.
Method: T4-like coliphages or a commercial Russian coliphage product or placebo was orally given over 4 days to Bangladeshi children hospitalized with acute bacterial diarrhea. Safety of oral phage was assessed clinically and by functional tests; coliphage and Escherichia coli titers and enteropathogens were determined in stool and quantitative diarrhea parameters (stool output, stool frequency) were measured. Stool microbiota was studied by 16S rRNA gene sequencing; the genomes of four fecal Streptococcus isolates were sequenced.
Findings: No adverse events attributable to oral phage application were observed (primary safety outcome). Fecal coliphage was increased in treated over control children, but the titers did not show substantial intestinal phage replication (secondary microbiology outcome). 60% of the children suffered from a microbiologically proven E. coli diarrhea; the most frequent diagnosis was ETEC infections. Bacterial co-pathogens were also detected. Half of the patients contained phage-susceptible E. coli colonies in the stool. E. coli represented less than 5% of fecal bacteria. Stool ETEC titers showed only a short-lived peak and were otherwise close to the replication threshold determined for T4 phage in vitro. An interim analysis after the enrollment of 120 patients showed no amelioration in quantitative diarrhea parameter by PT over standard care (tertiary clinical outcome). Stool microbiota was characterized by an overgrowth with Streptococcus belonging to the Streptococcus gallolyticus and Streptococcus salivarius species groups, their abundance correlated with quantitative diarrhea outcome, but genome sequencing did not identify virulence genes.
Interpretation: Oral coliphages showed a safe gut transit in children, but failed to achieve intestinal amplification and to improve diarrhea outcome, possibly due to insufficient phage coverage and too low E. coli pathogen titers requiring higher oral phage doses. More knowledge is needed on in vivo phage-bacterium interaction and the role of E. coli in childhood diarrhea for successful PT.
Funding: The study was supported by a grant from Nestlé Nutrition and Nestlé Health Science. The trial was registered with Identifier NCT00937274 at ClinicalTrials.gov.
Keywords: Bacteriophages; Bangladesh; Bifidobacterium; Cfu, colony forming unit; Children; Diarrhea; EAEC, enteroaggregative E. coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli; Escherichia coli; M, ColiProteus phage cocktail from Microgen; ORS, oral rehydration solution; P, placebo; PT, phage therapy; RCT, randomized controlled trial; Streptococcus; T, T4 phage cocktail from NRC; pfu, plaque forming unit; qPCR, quantitative polymerase chain reaction.
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References
- Ahmed T., Bhuiyan T.R., Zaman K., Sinclair D., Qadri F. Vaccines for preventing enterotoxigenic Escherichia coli (ETEC) diarrhoea. Cochrane Database Syst. Rev. 2013;7
- Albert M.J., Faruque A.S., Faruque S.M., Sack R.B., Mahalanabis D. Case–control study of enteropathogens associated with childhood diarrhea in Dhaka, Bangladesh. J. Clin. Microbiol. 1999;37:3458–3464.
- Allen H.K., Looft T., Bayles D.O. Antibiotics in feed induce prophages in swine fecal microbiomes. MBio. 2011;2
- Altschul S.F., Gish W., Miller W. Basic local alignment search tool. J. Mol. Biol. 1990;215:403–410.
- Barletta F., Ochoa T.J., Mercado E. Quantitative real-time polymerase chain reaction for enteropathogenic Escherichia coli: a tool for investigation of asymptomatic versus symptomatic infections. Clin. Infect. Dis. 2011;53:1223–1229.
- Boleij A., Tjalsma H. The itinerary of Streptococcus gallolyticus infection in patients with colonic malignant disease. Lancet Infect. Dis. 2013;13(8):719–724.
- Booijink C.C. High temporal and inter-individual variation detected in the human ileal microbiota. Environ. Microbiol. 2010;12(12):3213–3227.
- Bourdin G., Navarro A., Sarker S.A. Coverage of diarrhoea-associated Escherichia coli isolates from different origins with two types of phage cocktails. Microb. Biotechnol. 2014;7:165–176.
- Bourdin G., Schmitt B., Marvin Guy L. Amplification and purification of T4-like Escherichia coli phages for phage therapy: from laboratory to pilot scale. Appl. Environ. Microbiol. 2014;80:1469–1476.
- Brüssow H. Phage therapy: the Escherichia coli experience. Microbiology. 2005;151:2133–2140.
- Brüssow H. What is needed for phage therapy to become a reality in Western medicine? Virology. 2012;434:138–142.
- Brüssow H. Bacteriophage–host interaction: from splendid isolation into a messy reality. Curr. Opin. Microbiol. 2013;16:500–506.
- Bruttin A., Brüssow H. Human volunteers receiving Escherichia coli phage T4 orally: a safety test of phage therapy. Antimicrob. Agents Chemother. 2005;49:2874–2878.
- Caporaso J.G. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods. 2010;7:335–336.
- Casswall T.H., Sarker S.A., Faruque S.M. Treatment of enterotoxigenic and enteropathogenic Escherichia coli-induced diarrhoea in children with bovine immunoglobulin milk concentrate from hyperimmunized cows: a double-blind, placebo-controlled, clinical trial. Scand. J. Gastroenterol. 2000;35:711–718.
- Chen L., Xiong Z., Sun L., Yang J., Jin Q. VFDB 2012 update: toward the genetic diversity and molecular evolution of bacterial virulence factors. Nucleic Acids Res. 2012;40(Database issue):D641–D645.
- Chibani-Chennoufi S., Sidoti J., Bruttin A. Isolation of Escherichia coli bacteriophages from the stool of pediatric diarrhea patients in Bangladesh. J. Bacteriol. 2004;186:8287–8294.
- Chin C.S. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat. Methods. 2013;10(6):563–569.
- Collins J.W., Keeney K.M., Crepin V.F. Citrobacter rodentium: infection, inflammation and the microbiota. Nat. Rev. Microbiol. 2014;12:612–623.
- Danne C. Molecular characterization of a Streptococcus gallolyticus genomic island encoding a pilus involved in endocarditis. J. Infect. Dis. 2011;204:1960–1970.
- David L.A. Gut microbial succession follows acute secretory diarrhea in humans. MBio. 2015;6(3)
- Denou E., Bruttin A., Barretto C., Ngom-Bru C., Brüssow H., Zuber S. T4 phages against Escherichia coli diarrhea: potential and problems. Virology. 2009;388:21–30.
- Ding T., Schloss P.D. Dynamics and associations of microbial community types across the human body. Nature. 2014;509:357–360.
- Edgar R.C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26:2460–2461.
- Edgar R.C., Haas B.J., Clemente J.C., Quince C., Knight R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 2011;27:2194–2200.
- Fairbrother J.M., Nadeau E., Gyles C.L. Escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies. Anim. Health Res. Rev. 2005;6:17–39.
- Gardner S.N., Hall B.G. When whole-genome alignments just won't work: kSNP v2 software for alignment-free SNP discovery and phylogenetics of hundreds of microbial genomes. PLoS One. 2013;8(12):e81760. (2013 Dec 9)
- Goldsmith C.S., Miller S.E. Modern use of electron microscopy for detection of viruses. Clin. Microbiol. Rev. 2009;22:552–563.
- Guion C.E., Ochoa T.J., Walker C.M., Barletta F., Cleary T.G. Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR. J. Clin. Microbiol. 2008;46:1752–1757.
- Guzmán C., Mocé-Llivina L., Lucena F., Jofre J. Evaluation of Escherichia coli host strain CB390 for simultaneous detection of somatic and F-specific coliphages. Appl. Environ. Microbiol. 2008;74:531–534.
- Harris A.M., Chowdhury F., Begum Y.A. Shifting prevalence of major diarrheal pathogens in patients seeking hospital care during floods in 1998, 2004, and 2007 in Dhaka, Bangladesh. Am.J.Trop. Med. Hyg. 2008;79:708–714.
- Hilpert H., Brüssow H., Mietens C., Sidoti J., Lerner L., Werchau H. Use of bovine milk concentrate containing antibody to rotavirus to treat rotavirus gastroenteritis in infants. J. Infect. Dis. 1987;156:158–166.
- Holmes I., Harris K., Quince C. Dirichlet multinomial mixtures: generative models for microbial metagenomics. PLoS One. 2012;7
- Hsiao A., Ahmed A.M., Subramanian S. Members of the human gut microbiota involved in recovery from Vibrio cholerae infection. Nature. 2014;515:423–426.
- Jiang Z.D., Lowe B., Verenkar M.P. Prevalence of enteric pathogens among international travelers with diarrhea acquired in Kenya (Mombasa), India (Goa), or Jamaica (Montego Bay) J. Infect. Dis. 2002;185:497–502.
- Jin D. Dynamics of fecal microbial communities in children with diarrhea of unknown etiology and genomic analysis of associated Streptococcus lutetiensis. BMC Microbiol. 2013;13
- Junick J., Blaut M. Quantification of human fecal bifidobacterium species by use of quantitative real-time PCR analysis targeting the groEL gene. Appl. Environ. Microbiol. 2012;78:2613–2622.
- Kotloff K.L., Nataro J.P., Blackwelder W.C. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case–control study. Lancet. 2013;382:209–222.
- Liu B., Pop M. ARDB—antibiotic resistance genes database. Nucleic Acids Res. 2009;37(Database issue):D443–D447. (2009 Jan)
- Liu J., Kabir F., Manneh J. Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study. Lancet Infect. Dis. 2014;14:716–724.
- Martinez L. Options for Action. WHO; Geneva: 2012. The evolving threat of antimicrobial resistance.
- McCallin S., Sarker S.A., Barretto C. Safety analysis of a Russian phage cocktail: from MetaGenomic analysis to oral application in healthy human subjects. Virology. 2013;443:187–196.
- Międzybrodzki R., Borysowski J., Weber-Dąbrowska B. Clinical aspects of phage therapy. Adv. Virus Res. 2012;83:73–121.
- Nadkarni M.A., Martin F.E., Jacques N.A., Hunter N. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology. 2002;148:257–266.
- Piddock L.J. The crisis of no new antibiotics—what is the way forward? Lancet Infect. Dis. 2012;12:249–253.
- Pop M. Diarrhea in young children from low-income countries leads to large-scale alterations in intestinal microbiota composition. Genome Biol. 2014;15(6):R76.
- Qadri F., Svennerholm A.M., Faruque A.S., Sack R.B. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin. Microbiol. Rev. 2005;18:465–483.
- Quince C. Accurate determination of microbial diversity from 454 pyrosequencing data. Nat. Methods. 2009;6:639–641.
- Sanchez M., Darimont C., Drapeau V. Effect of Lactobacillus rhamnosus CGMCC1.3724 supplementation on weight loss and maintenance in obese men and women. Br. J. Nutr. 2014;111:1507–1519.
- Sarker S.A., Casswall T.H., Mahalanabis D. Successful treatment of rotavirus diarrhea in children with immunoglobulin from immunized bovine colostrum. Pediatr. Infect. Dis. J. 1998;17:1149–1154.
- Sarker S.A., McCallin S., Barretto C. Oral T4-like phage cocktail application to healthy adult volunteers from Bangladesh. Virology. 2012;434:222–232.
- Sarker S.A., Sultana S., Fuchs G.J. Lactobacillus paracasei strain ST11 has no effect on rotavirus but ameliorates the outcome of nonrotavirus diarrhea in children from Bangladesh. Pediatrics. 2005;116:e221–e228.
- Schlegel L., Grimont F., Ageron E., Grimont P.A., Bouvet A. Reappraisal of the taxonomy of the Streptococcus bovis/Streptococcus equinus complex and related species: description of Streptococcus gallolyticus subsp. gallolyticus subsp. nov., S. gallolyticus subsp. macedonicus subsp. nov. and S. gallolyticus subsp. pasteurianus subsp. nov. Int. J. Syst. Evol. Microbiol. 2003;53(Pt 3):631–645.
- Schloss P.D., Westcott S.L. Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis. Appl. Environ. Microbiol. 2011;77:3219–3226.
- Schloss P.D. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 2009;75:7537–7541.
- Simeoni U., Berger B., Junick J. Gut microbiota analysis reveals a marked shift to bifidobacteria by a starter infant formula containing a synbiotic of bovine milk-derived oligosaccharides and Bifidobacterium animalis subsp. lactis CNCM I-3446. Environ. Microbiol. 2015 (Epub ahead of print)
- Stanton T.B. A call for antibiotic alternatives research. Trends Microbiol. 2013;21:111–113.
- Sulakvelidze A., Alavidze Z., Morris J.G., Jr. Bacteriophage therapy. Antimicrob. Agents Chemother. 2001;45:649–659.
- Svenungsson B., Lagergren A., Ekwall E. Enteropathogens in adult patients with diarrhea and healthy control subjects: a 1-year prospective study in a Swedish clinic for infectious diseases. Clin. Infect. Dis. 2000;30:770–778.
- Taniuchi M., Sobuz S.U., Begum S. Etiology of diarrhea in Bangladeshi infants in the first year of life analyzed using molecular methods. J. Infect. Dis. 2013;208:1794–1802.
- Van den Bogert B., Boekhorst J., Herrmann R., Smid E.J., Zoetendal E.G., Kleerebezem M. Comparative genomics analysis of Streptococcus isolates from the human small intestine reveals their adaptation to a highly dynamic ecosystem. PLoS One. 2013;8(12)
- Vidal M., Kruger E., Durán C. Single multiplex PCR assay to identify simultaneously the six categories of diarrheagenic Escherichia coli associated with enteric infections. J. Clin. Microbiol. 2005;43:5362–5365.
- Weiss M., Denou E., Bruttin A., Serra-Moreno R., Dillmann M.L., Brüssow H. In vivo replication of T4 and T7 bacteriophages in germ-free mice colonized with Escherichia coli. Virology. 2009;393:16–23.
- Wiggins B.A., Alexander M. Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems. Appl. Environ. Microbiol. 1985;49:19–23.
- Wright A., Hawkins C.H., Anggård E.E., Harper D.R. A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic-resistant Pseudomonas aeruginosa; a preliminary report of efficacy. Clin. Otolaryngol. 2009;34:349–357.
- Yang C.J., Lee N.Y., Lin Y.H. Jarisch–Herxheimer reaction after penicillin therapy among patients with syphilis in the era of the HIV infection epidemic: incidence and risk factors. Clin. Infect. Dis. 2010;51:976–979.
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