Oral Phage Therapy of Acute Bacterial Diarrhea With Two Coliphage Preparations: A Randomized Trial in Children From Bangladesh.

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.

Coverage of diarrhoea-associated Escherichia coli isolates from different origins with two types of phage cocktails.

Eighty-nine T4-like phages from our phage collection were tested against four collections of childhood diarrhoea-associated Escherichia coli isolates representing different geographical origins (Mexico versus Bangladesh), serotypes (69 O, 27 H serotypes), pathotypes (ETEC, EPEC, EIEC, EAEC, VTEC, Shigella), epidemiological settings (community and hospitalized diarrhoea) and years of isolation. With a cocktail consisting of 3 to 14 T4-like phages, we achieved 54% to 69% coverage against predominantly EPEC isolates from Mexico, 30% to 53% against mostly ETEC isolates from a prospective survey in Bangladesh, 24% to 61% against a mixture of pathotypes isolated from hospitalized children in Bangladesh, and 60% coverage against Shigella isolates. In comparison a commercial Russian phage cocktail containing a complex mixture of many different genera of coliphages showed 19%, 33%, 50% and 90% coverage, respectively, against the four above-mentioned collections. Few O serotype-specific phages and no broad-host range phages were detected in our T4-like phage collection. Interference phenomena between the phage isolates were observed when constituting larger phage cocktails. Since the coverage of a given T4-like phage cocktail differed with geographical area and epidemiological setting, a phage composition adapted to a local situation is needed for phage therapy approaches against E. coli pathogens.

Amplification and purification of T4-like escherichia coli phages for phage therapy: from laboratory to pilot scale.

We investigated the amplification and purification of phage preparations with respect to titer, contamination level, stability, and technical affordability. Using various production systems (wave bags, stirred-tank reactors, and Erlenmeyer flasks), we obtained peak titers of 10(9) to 10(10) PFU/ml for T4-like coliphages. Phage lysates could be sterilized through 0.22-µm membrane filters without titer loss. Phages concentrated by differential centrifugation were not contaminated with cellular debris or bacterial proteins, as assessed by electron microscopy and mass spectrometry, respectively. Titer losses occurred by high-speed pelleting of phages but could be decreased by sedimentation through a sucrose cushion. Alternative phage concentration methods are prolonged medium-speed centrifugation, strong anion-exchange chromatography, and ultrafiltration, but the latter still allowed elevated lipopolysaccharide contamination. T4-like phages could not be pasteurized but maintained their infectivity titer in the cold chain. In the presence of 10 mM magnesium ions, phages showed no loss of titer over 1 month at 30°C.

Safety analysis of a Russian phage cocktail: from metagenomic analysis to oral application in healthy human subjects.

Phage therapy has a long tradition in Eastern Europe, where preparations are comprised of complex phage cocktails whose compositions have not been described. We investigated the composition of a phage cocktail from the Russian pharmaceutical company Microgen targeting Escherichia coli/Proteus infections. Electron microscopy identified six phage types, with numerically T7-like phages dominating over T4-like phages. A metagenomic approach using taxonomical classification, reference mapping and de novo assembly identified 18 distinct phage types, including 7 genera of Podoviridae, 2 established and 2 proposed genera of Myoviridae, and 2 genera of Siphoviridae. De novo assembly yielded 7 contigs greater than 30 kb, including a 147-kb Myovirus genome and a 42-kb genome of a potentially new phage. Bioinformatic analysis did not reveal undesired genes and a small human volunteer trial did not associate adverse effects with oral phage exposure.

Oral T4-like phage cocktail application to healthy adult volunteers from Bangladesh.

The genomic diversity of 99 T4-like coliphages was investigated by sequencing an equimolar mixture with Illumina technology and screening them against different databases for horizontal gene transfer and undesired genes. A 9-phage cocktail was given to 15 healthy adults from Bangladesh at a dose of 3×10(9) and 3×10(7) plaque-forming units and placebo respectively. Phages were detected in 64% of the stool samples when subjects were treated with higher titer phage, compared to 30% and 28% with lower-titer phage and placebo, respectively. No Escherichia coli was present in initial stool samples, and no amplification of phage was observed. One percent of the administered oral phage was recovered from the feces. No adverse events were observed by self-report, clinical examination, or from laboratory tests for liver, kidney, and hematology function. No impact of oral phage was seen on the fecal microbiota composition with respect to bacterial 16S rRNA from stool.

Tolerance, safety, and effect on the faecal microbiota of an enteral formula supplemented with pre- and probiotics in critically ill children.

OBJECTIVES: The aim of this study was to demonstrate the tolerance and safety of an enteral formula containing prebiotics/probiotics, and its effect on the faecal microbiota in critically ill children. SUBJECTS AND METHODS: Ninety-four patients between 1 and 3 years old under mechanical ventilation requiring enteral feeding were randomised to receive either a test formula containing a synbiotic blend (composed of 2 probiotic strains [Lactobacillus paracasei NCC 2461 and Bifidobacterium longum NCC 3001], fructooligosaccharides [FOS], inulin, and Acacia gum), or a control formula. Patients remained in the intensive care unit for 7 days and were examined at day 14. Tolerance was assessed by overall caloric intake and time to reach caloric goal. Safety was assessed by abdominal distention, vomiting, and stool frequency. Microbiota was analysed by culture- and molecular-based methods. RESULTS: Overall caloric intake and time to reach caloric goal were similar between groups (noninferiority was shown). Abdominal distention, vomiting, and stool frequency were not affected by the supplementation with pre- and probiotics. Faecal bifidobacteria were higher in the test group at the end of the study. A similar trend was observed for total lactobacilli. L paracasei NCC 2461 and B longum NCC 3001 were detected in 80.4% and 17% of the test group patients, respectively. Enterobacteria levels remained unchanged during hospitalisation in the control group but diminished in the test group. CONCLUSIONS: The enteral formula supplemented with synbiotics was well tolerated by children in intensive care units; it was safe and produced an increase in faecal bacterial groups of previously reported beneficial effects.

Bifidobacterium lactis attenuates onset of inflammation in a murine model of colitis.

AIM: To assess the anti-inflammatory effect of the probiotic Bifidobacterium lactis (B. lactis) in an adoptive transfer model of colitis. METHODS: Donor and recipient mice received either B. lactis or bacterial culture medium as control (deMan Rogosa Sharpe) in drinking water for one week prior to transfer of a mix of naive and regulatory T cells until sacrifice. RESULTS: All recipient mice developed signs of colonic inflammation, but a significant reduction of weight loss was observed in B. lactis-fed recipient mice compared to control mice. Moreover, a trend toward a diminution of mucosal thickness and attenuated epithelial damage was revealed. Colonic expression of pro-inflammatory and T cell markers was significantly reduced in B. lactis-fed recipient mice compared to controls. Concomitantly, forkhead box protein 3, a marker of regulatory T cells, was significantly up-regulated by B. lactis. CONCLUSION: Daily oral administration of B. lactis was able to reduce inflammatory and T cells mediators and to promote regulatory T cells specific markers in a mouse model of colitis.

Effect of Lactobacillus paracasei NCC2461 on antigen-specific T-cell mediated immune responses in aged mice.

Aging is associated with a reduced capacity to mount proper immune responses, in particular to vaccines. Probiotic lactic acid bacteria may improve the immune status of the elderly; however, there is little evidence showing an effect of these bacteria on humoral and cellular immune responses. In the present study, the immunomodulatory capacity of the probiotic Lactobacillus paracasei NCC2461 combined or not with a prebiotic composition, FOS/inulin, was examined in aged mice. Male C57BL/6J mice (21-months-old) were allocated to one of three groups fed ad libitum for 44 days with different diets: a normal diet (control), a normal diet plus NCC2461 given in the drinking water, or a diet containing FOS/inulin plus NCC2461 in the drinking water. All mice were immunized on day 15 and challenged on day 22 with keyhole limpet hemocyanin (KLH). T helper (Th)1 cell-dependent immune responses (anti-KLH immunoglobulin G(2a) [IgG(2a)] levels and delayed type hypersensitivity response) were increased significantly in NCC2461-supplemented mice when compared to controls. Supplementation with FOS/inulin did not further improve the immune-enhancing effect mediated by the probiotic. Splenocyte proliferation, T cell subsets, systemic total IgG levels, and mucosal total IgA responses were not affected. Interestingly, supplementation with NCC2461 modulated the intestinal microbiota composition by increasing the numbers of bifidobacteria and lactobacilli. In conclusion, oral intake of L. paracasei NCC2461 by aged mice enhanced the specific adaptive immune response to in vivo antigenic challenge without altering other cellular and humoral immune responses. The poor responsiveness to antigenic challenge, frequently observed in elderly people, may be improved by supplementation with L. paracasei NCC2461.