In vitro and in vivo bacteriolytic activities of Escherichia coli phages: implications for phage therapy

Abstract

Four T4-like coliphages with broad host ranges for diarrhea-associated Escherichia coli serotypes were isolated from stool specimens from pediatric diarrhea patients and from environmental water samples. All four phages showed a highly efficient gastrointestinal passage in adult mice when added to drinking water. Viable phages were recovered from the feces in a dose-dependent way. The minimal oral dose for consistent fecal recovery was as low as 10(3) PFU of phage per ml of drinking water. In conventional mice, the orally applied phage remained restricted to the gut lumen, and as expected for a noninvasive phage, no histopathological changes of the gut mucosa were detected in the phage-exposed animals. E. coli strains recently introduced into the intestines of conventional mice and traced as ampicillin-resistant colonies were efficiently lysed in vivo by phage added to the drinking water. Likewise, an in vitro phage-susceptible E. coli strain freshly inoculated into axenic mice was lysed in vivo by an orally applied phage, while an in vitro-resistant E. coli strain was not lysed. In contrast, the normal E. coli gut flora of conventional mice was only minimally affected by oral phage application despite the fact that in vitro the majority of the murine intestinal E. coli colonies were susceptible to the given phage cocktail. Apparently, the resident E. coli gut flora is physically or physiologically protected against phage infection.

FIG. 1.

Four T4-like phages used in the mouse experiments. (A) Transmission electron microscopy picture of CsCl density gradient-purified bacteriophage JS4 (a), JSD.1 (b), JSL.6 (c), and JS94.1 (d). Negative staining was performed with uranyl acetate (c), ammonium molybdate (a and d), or phosphotungstic acid (c). The size bar corresponds to 100 nm. (B) Restriction analysis of phages (for lanes a to d, see corresponding subpanel in panel A; lane e, phage T4) with enzyme DraI. Lane M, DNA size marker (1-kb lambda DNA ladder; Invitrogen).

FIG. 2.

Lysis of E. coli K803 strain by the four T4-like phages in broth culture. (A) OD development of an uninfected control culture (K-12) and parallel cultures infected with phages JS94.1, JS4, JSD.1, and JSL.6. (B) Progeny phage release from the four phage-infected cultures depicted in panel A. Phage infectivity was measured by plaque assay.

FIG. 3.

Gastrointestinal passage of the orally added phages in conventional mice. Fecal phage titer after oral addition of the specified phage strain at 10⁶ (circles), 10⁵ (diamonds), 10⁴ (squares), and 10³ (bars) PFU/ml, fed to four mice at the times indicated by the shaded bars at the bottom of the figure. The triangles give the phage titers for the control mice. The periods of phage-free drinking water are indicated by white boxes.

FIG. 4.

Effect of oral phage on the inoculated E. coli strain in axenic mice. (A) Fecal E. coli counts (solid line in log CFU per milliliter) and fecal phage counts (dashed line in log PFU per milliliter) in two axenic mice exposed to the specified E. coli strains, phage JS94.1, or water at the specified time points; the start day is indicated with an arrow below the abscissa. A black line with squares or a gray line with triangles identifies values from an individual mouse. (B) 10⁶ PFU/ml was given from day 1 in the drinking water to axenic mice lacking intestinal bacteria. The mice were force-fed with 10⁴ CFU of K803 at day 8. Logarithmic fecal cell (solid line) and phage counts (dashed line) per gram of stool were plotted over 10 days. A black line with squares or a gray line with triangles identifies values from an individual mouse.

FIG. 5.

Effect of oral phage on the introduction of ampicillin-resistant E. coli in mice. (A) Fecal cell counts in three mice force-fed with 5 × 10⁷ CFU of ampicillin-resistant E. coli and ampicillin (A/E) at the time points marked with an arrow below the time axis. The ordinate shows the logarithm of CFU per gram of stool. Each vertical bar represents the fecal cell count for one animal at the specified time point. (B) The same experiment as depicted in panel A except that in addition to ampicillin the mice also received the phage cocktail at 10⁶ PFU/ml in the drinking water. The rightmost black data points refer to a control mouse not receiving ampicillin-resistant E. coli.

FIG. 6.

Effect of oral phage on the introduction of ampicillin-resistant E. coli in mice pretreated with ampicillin in the drinking water. (A) Seven mice received ampicillin by force-feeding at day 1 and in the drinking water throughout the experiment. At the time points indicated with arrows marked with A/E, six mice were force-fed with ampicillin and ampicillin-resistant E. coli (a control mouse received only buffer instead of E. coli). Both groups of mice received ampicillin in the drinking water, but some mice were in addition exposed to 10⁶ PFU/ml of the phage cocktail in the drinking water (B). Both panels show the fecal counts of ampicillin-resistant cells. The rightmost black data points in panel B refer to a control mouse not receiving ampicillin-resistant E. coli.