Isolation of Escherichia coli bacteriophages from the stool of pediatric diarrhea patients in Bangladesh

Abstract

A 3-week coliphage survey was conducted in stool samples from 140 Bangladeshi children hospitalized with severe diarrhea. On the Escherichia coli indicator strain K803, all but one phage isolate had 170-kb genomes and the morphology of T4 phage. In spot tests, the individual T4-like phages infected up to 27 out of 40 diarrhea-associated E. coli, representing 22 O serotypes and various virulence factors; only five of them were not infected by any of these new phages. A combination of diagnostic PCR based on g32 (DNA binding) and g23 (major capsid protein) and Southern hybridization revealed that half were T-even phages sensu strictu, while the other half were pseudo-T-even or even more distantly related T4-like phages that failed to cross-hybridize with T4 or between each other. Nineteen percent of the acute stool samples yielded T4-like phages, and the prevalence was lower in convalescent stool samples. T4-like phages were also isolated from environmental and sewage water, but with low frequency and low titers. On the enteropathogenic E. coli strain O127:K63, 14% of the patients yielded phage, all of which were members of the phage family Siphoviridae with 50-kb genomes, showing the morphology of Jersey- and beta-4 like phages and narrow lytic patterns on E. coli O serotypes. Three siphovirus types could be differentiated by lack of cross-hybridization. Only a few stool samples were positive on both indicator strains. Phages with closely related restriction patterns and, in the case of T4-like phages, identical g23 gene sequences were isolated from different patients, suggesting epidemiological links between the patients.

FIG. 1.

Genome size and morphology of the phages isolated from the stools of pediatric diarrhea patients. (A) Transmission electron microscopy picture of CsCl density gradient-purified myophages JS98 (a) and JSL.3 (b). Negative staining was with uranyl acetate. The size bar correspond to 100 nm. (B) Pulsed-field gel electrophoresis of myophages (lanes a, c, d, and e; lane b, T4 phage control) and of siphophages (lanes f to i) isolated from the stools of different patients. All isolates are from different patients lane a, JS4; lane 6, JS94.1; lane d, JSD.1; lane e, JS94.3; lane f, JS83.1; lane g, JS20; lane h, JS77.2; lane i, JS122.1. M, size markers (phage lambda concatemers, 50-kb DNA ladder; Promega). (C) Transmission electron microscopy picture of CsCl density gradient-purified bacteriophage JS77.1 after negative staining with uranyl acetate (a), phage JS83.2 after negative staining with phosphotungstic acid (b), and phage JS61.2 (c) and phage JS122.1 (d) after negative staining with ammonium molybdate coupled with bacitracin. The bar corresponds to 100 nm.

FIG. 2.

Restriction analysis of siphophages with 50-kb genomes. EcoRV restriction digests of phages isolated from stool samples of eight different patients identified with their JS code number at the top of the lanes. M, size markers, 1-kb ladder.

FIG. 3.

Restriction and Southern analysis of the stool and environmental water myophages with 170-kb genomes. A. PacI digests of five stool myophages from different children identified with their JS code number, seven environmental water myophages identified with their geographical origin (D, Dhaka; L, Lausanne) at the top of the lanes, and two reference phages (RB33 and T4). M, 1-kb lambda DNA ladder. B and C. Corresponding Southern blot hybridization with radiolabeled phage T4 DNA (B) or phage JSL.5 (C) as a probe.

FIG. 4.

PCR analysis of the phages with a 170-kb genome. A. PCR with primers FR60 and FR61 amplifying gene 32 from stool samples. Gel analysis of the PCR fragments obtained with heat-treated phage particles from eight different stool phage isolates derived from seven different patients identified by their patient number at the top of the lanes. The lane marked with + is the positive control with T4 phage DNA and − is the negative control containing the master mix without DNA. M, 1-kb lambda DNA ladder. B. PCR with primers FR60 and FR61 amplifying gene 32 from four phages isolated from environmental water samples identified by their geographical origin at the top of the lanes. RB33 and T4 are the positive controls. 1 and 4 are negative controls with stool phages JS1 and JS4. C. PCR with primers Mzia1 and CAP8 amplifying gene 23 with heat-treated phage particles from eight stool isolates of seven different patients identified by their code number, two water phages identified by their geographical origin, T4 phage as a positive control, and no phage particles as the negative control (−). M, 1-kb lambda DNA ladder.