In Vitro and In Vivo Bacteriolytic Activities

of Escherichia coli Phages: Implications

for Phage Therapy

Sandra Chibani-Chennoufi, Josette Sidoti, Anne Bruttin,

Elizabeth Kutter, Shafiq Sarker and Harald Br ssow

Antimicrob. Agents Chemother. 2004, 48(7):2558. DOI:

10.1128/AAC.48.7.2558-2569.2004.

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2004, p. 2558 2569 Vol. 48, No. 7

0066-4804/04/$08.00 0 DOI: 10.1128/AAC.48.7.2558 2569.2004

Copyright 2004, American Society for Microbiology. All Rights Reserved.

In Vitro and In Vivo Bacteriolytic Activities of Escherichia coli Phages:

Implications for Phage Therapy

Sandra Chibani-Chennou,1 Josette Sidoti,1 Anne Bruttin,1 Elizabeth Kutter,2

Sha q Sarker,3 and Harald Brussow1*

Nestle Research Center, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland1; The Evergreen State College,

Olympia, Washington 985052; and ICDDR,B: Centre for Health and Population Research, Dhaka 1000

Mohakhali, Dhaka 1212, Bangladesh3

Received 21 July 2003/Returned for modi cation 4 November 2003/Accepted 26 February 2004

Four T4-like coliphages with broad host ranges for diarrhea-associated Escherichia coli serotypes were

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isolated from stool specimens from pediatric diarrhea patients and from environmental water samples. All four

phages showed a highly ef cient 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 103 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 ef ciently 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 ora 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 ora is physically or physiologically protected against phage infection.

An ideal candidate for phage therapy of E. coli infections is the

Diarrhea is the second most common cause of morbidity and

mortality among infants and children in developing countries, coliphage T4 family. T4 is arguably the best-characterized biolog-

exceeded only by respiratory diseases (47). Diarrhea has a ical system (25). This phage family is a natural component of the

complex etiology (22). However, Escherichia coli and rotavirus mammalian gut and can be easily isolated from the environment

account for up to 50% of childhood diarrhea cases in devel- (stool and sewage) (1, 20, 21, 29). The richest sources of T4-like

oping countries (2, 3, 16, 23, 26). Enterotoxigenic E. coli phages are apparently stools of diarrhea patients (20, 21). T4-like

phages can be grown to high titer on laboratory E. coli strains.

(ETEC) is also a leading cause of traveler s diarrhea (9). Al-

though the use of oral rehydration solutions has substantially Early during the infection cycle T4 degrades the host DNA to the

reduced mortality from dehydration, it has little or no effect on nucleotide level, preventing any integration of phage DNA into

the diarrhea itself and no effect on the transmission of the the bacterial chromosome (lysogeny) (34). T4 and a number of

disease (7). Vaccines against diarrhea-causing E. coli are still in related phages have been completely sequenced (http://phage-

an early developmental stage (42, 43). A potentially low-cost .bioc.tulane.edu/) (33), and no phage-associated bacterial viru-

treatment option for bacterial diarrhea was proposed 80 years lence factors have been detected in these phages.

ago by Felix d Herelle in the form of bacterial viruses (bacte-

Despite these assets of the T4 phage system, the potential of

riophages). Indeed, in the 1930s American physicians used bacteriophage therapy in human infections has not yet been care-

pharmaceutical phage preparations for the treatment of both fully documented in scienti c publications. Part of the academic

diarrheal diseases and staphylococcal infections (reviewed in community is therefore still phage skeptic. They point to the

references 15 and 50). The development of antibiotics in the experience that the tremendous in vitro lytic activities of coliph-

1940s replaced phages as therapeutic agents in the West, al- ages was rarely, if ever, demonstrated in carefully documented in

though enteric diseases, nosocomial infections, burns and vivo situations. In fact, not much is known about the factors

wound infections continued to be treated with phage prepara- governing the phage-bacterium interaction in the context of the

tions in the Soviet Union on a very large scale (49). Western complex microbial environment of the mammalian gut. To help

scientists were either unaware of this work or remained dubi- ll this gap, we report here on the gastrointestinal passage of a set

ous about the reported high success rate despite the positive of orally applied T4-like phages in mice. In addition, we studied

results obtained with the phage treatment of E. coli infections their in vivo bacteriolytic activities on the resident gut E. coli ora

and towards E. coli strains introduced into the gut.

in a number of farm animals (calves, piglets, lambs, and chick-

ens) (6, 45, 46).

MATERIALS AND METHODS

Phage isolation. Phage JS4 and JS94.1 were isolated from stool samples of

* Corresponding author. Mailing address: Nestle Research Center,

pediatric patients with undifferentiated diarrhea hospitalized at the International

Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland. Center for Diarrheal Disease Research in Dhaka, Bangladesh. The stool sample

Phone: 41-21-785-**-**. Fax: 41-21-785-**-**. E-mail: harald ( 10g) was resuspended in TS (NaCl [8.5 g/liter], tryptone [1 g/liter]) to a nal

.abqndu@r.postjobfree.com. volume of 30 ml and centrifuged for 15 min at 14,500 g in 50-ml Falcon tubes.

2558

VOL. 48, 2004 BACTERIOLYTIC ACTIVITIES OF E. COLI PHAGES 2559

One milliliter of each stool preparation was ltered through a Millex AP20 ml of phage buffer (20 mM Tris-HCl [pH 7.4], 100 mM NaCl, 10 mM MgSO4),

pre lter followed by a 0.45- m-pore-size Minisart lter. Subsequently, the sam- loaded on a discontinuous CsCl gradient (CsCl at 1.35, 1.53, and 1.65 g/ml), and

ples were stored at 4 C. Phage JSD.1 was isolated from environmental water in centrifuged at 4 C in an SW55 rotor at 40,000 rpm for 3 h in a Beckman L8-60

Dhaka, Bangladesh, and phage JSL.6 was isolated from a sewage station in Vidy M ultracentrifuge. Puri ed phage were recovered with a syringe and dialyzed

(Lausanne), Switzerland. Fifty milliliters of water samples was centrifuged at against phage buffer.

10,000 rpm for 15 min and ltered through a 0.45- m-pore-size Minisart lter. Electron microscopy. A drop of the phage suspension was applied to a Form-

The presence of phages was screened on the laboratory strain K803 (a K-12 var carbon-coated copper grid for 5 min; the suspension was removed with a

derivative lacking prophage lambda, described in reference 5). The strain lacks pipette and immediately replaced by a mixture of solutions A and B (solution A,

restriction-modi cation systems and prophage lambda. K-12 is one of the major 2% ammonium molybdate at pH 7.0 or 2% PTA; solution B, 11% bacitracin in

strains that have been widely used for phage studies as well as for recombinant distilled water) or a solution of 3% uranyl acetate. After 1 min the liquid was

DNA work. It is susceptible, for example, to nearly all of the over 100 T4-like removed with a lter paper. The grids were examined in a Philips CM12 trans-

phages in the Evergreen collection, most of which were isolated on E. coli B or mission electron microscope at 80 kV (magni cation, 176,000 or 224,000).

on some pathogenic strain. The K803 strain was propagated in Hershey broth The dimensions of the phage were calibrated with T4 phage particles (25).

(prepared according to the recipe in reference 26) at 37 C with agitation (240 DNA puri cation. Puri ed phages were treated with proteinase K at a nal

rpm). After overnight growth, the strains were streaked on a Hershey agar petri concentration of 1 mg/ml for 2 h at 37 C, and 3 M sodium acetate (pH 4.3) was

dish. Each time needed, a new culture was grown from a single colony. The stock added. DNA was extracted twice by phenol-chloroform and precipitated with 2

cultures were kept as stab cultures at 4 C. volumes of ethanol. After centrifugation, pellets were washed with 70% ethanol

Spot testing was done on Hershey plates (15 g/liter agar) overlaid with 3.5 ml and resuspended in 50 l of Tris-EDTA. DNA was digested with restriction

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of Hershey top agar (7.5 g/liter). Ten microliters of ltered samples was put as enzymes according to the instructions provided by the manufacturer.

eight spots in clockwise distribution around the plate after the top agar with Experimental animals. Eight-week-old C3H male mice (Charles River, St.

plating bacteria solidi ed. For phage plaque assays, top agar (7.5 g/liter) was Germain sur l Arbre, France) were held under standard animal house conditions

inoculated with 200 l of a fresh overnight culture and 100 l of positive sample and fed irradiated 03-40 chow from Usine d Alimentation Rationelle (Villem-

and incubated overnight at 37 C oissin-Orge, France). The drinking water, which did or did not contain phage at

One well-separated phage plaque was chosen for ampli cation from each the speci ed titer, consisted of Vittel mineral water. We initially used this water

positive stool sample, picked with a sterile toothpick, and inoculated into 5 ml of since it contained bicarbonate at 258 mg/liter, reasoning that bicarbonate would

Hershey broth together with 1% of an overnight culture of the E. coli strain buffer the stomach acidity and allow more ef cient stomach passage of the phage.

K803. Incubation was performed with agitation at 240 rpm at 37 C. When lysis However, in later experiments we observed that mineral waters containing less

occurred, 3 drops of chloroform was added. The lysate was left overnight at room bicarbonate (65 mg/liter) allowed an equally ef cient gut passage of the phage

temperature followed by centrifugation at 14,500 g for 10 min. The superna- (data not shown). The mineral water was changed every two days. Feces were

tant was transferred into a screw-cap glass tube. Three drops of fresh chloroform sampled once a day directly from the hand-held animal into a sterile tube by

was added, and the phage stock was stored at 4 C. The phage lysate was at least gently pressing the abdomen of the animals to avoid contamination of the stool

diluted 1,000-fold into mineral water for the mouse feeding experiments. with bedding material or dripping from the water bottles. For each experimental

Lysis in tube. The lysis test was done as follows. Five milliliters of Hershey series, ve mice were used. Each mouse was held in a Makrolon type 3 cage with

broth (23) was inoculated with 1% of a freshly grown culture (109 CFU/ml) and a ltered lid (Indulab, Gams, Switzerland), preventing cross-contamination be-

1% phage lysate (108 PFU/ml). Incubation at 37 C was continued under aerobic tween the cages. Stool samples were resuspended in 1 ml of phosphate-buffered

conditions in a shaking incubator (240 rpm) for 3 to 5 h until the uninfected saline. Tenfold dilutions of the resuspended feces were plated on Drigalski agar

control cells reached the stationary growth phase. The optical density (OD) in (Bio-Rad). This is a medium recommended for coproculture (Diagnostics Pas-

the phage-inoculated cell was compared to that of mock-infected control cells. teur). The medium is not speci c for E. coli but allows the differentiation of

For anaerobic conditions, tubes were held in an anaerobic jar at 37 C for 5 to lactose-fermenting colonies (E. coli, Klebsiella, Enterobacter) yielding yellow col-

10 h. The media were not prereduced; there was thus signi cant oxygen present onies from lactose-nonfermenters (Salmonella, Shigella, Proteus, Providencia,

during the early hours of the experiment. Hafnia, Serratia, Levinea, Edwardsiella, Alcaligenes, Pseudomonas) yielding blue-

Broth culture of phages. E. coli was inoculated 1:100 in 200 ml of Hershey green colonies (Diagnostics Pasteur). Practically all colonies from mouse fecal

medium and incubated at 37 C in a shaking incubator (240 rpm). When an OD pellets were lactose positive. Since Klebsiella and Enterobacter species do not

at 600 nm of 0.1 was reached, stock phages were inoculated with 107 PFU/ml. belong to the normal mouse fecal ora (52), the yellow colony count is practically

Each 15 min, samples were taken and OD readings of infected and uninfected an E. coli count. This diagnosis was con rmed by phage susceptibility: practically

cells were done at 600 nm. Samples were then centrifuged (10,000 g, 5 min, all colonies were lysed by one of the T4-like E. coli phages from our collection

20 C), and chloroform-treated phage was titrated in the supernatant. (see Results). We con rmed that the T4-like phages did not lyse a distinct genus

Pathogenic E. coli strains. The tested collection of pathogenic E. coli strains of Enterobacteriaceae, for example, the food pathogen Enterobacter sakazaki (P.

included 12 enteropathogenic E. coli (EPEC) strains, representing the major Breeuwer, unpublished results). The various T4-like phages were added to the

serotypes isolated worldwide from pediatric diarrhea patients (41). This set of drinking water in dilutions as speci ed in the text. In the experiments four mice

strains covered 10 different somatic O antigens and 10 different capsular K received mineral water with phage while one negative mouse control in each

antigens (Table 1). In addition, the collection contained 12 major ETEC sero- experiment received only mineral water. Phages were titrated by the plaque assay

types isolated from either pediatric gastroenteritis patients or adults suffering in ltered fecal samples on the E. coli indicator cell K803. At the end of the

from traveler s diarrhea (Table 1). The ETEC strains represented 11 further O experiment, the mice were sacri ced and standard gross anatomical and his-

antigens, 10 distinct H antigens, and various combinations of heat-stable (ST) topathological examinations were conducted. Different parts of the gut were

and heat-labile (LT) toxin producers (Table 1). These 22 pathogenic E. coli rinsed with physiological salt before cells and phages were counted by colony and

strains were obtained from B. Rowe (Central Public Health Laboratory, London, plaque assay.

United Kingdom). Twelve further distinct ETEC and six EPEC strains were Mouse experiments with ampicillin-resistant cells. E. coli K803 was grown in

obtained from the microbiology laboratory of the International Center for Di- Hershey broth to an OD (600 nm) of 0.7. Cells were centrifuged for 20 min at

arrheal Disease Research. They represent predominant E. coli isolates from their 4,500 rpm. The pellet was carefully resuspended in cold H2O (4 C). The cells

hospitalized pediatric diarrhea patients. The pathogenic E. coli strains from were then washed twice with cold 10% glycerol. Finally the pellet was resus-

Dhaka were typed by DNA probes for the presence of ST and LT enterotoxin, pended in 1 ml of cold glycerol and kept at 80 C as competent cells.

ST, colonization factor antigen (CFA), E. coli surface antigens (CS), and the One hundred microliters of cells was electroporated with 0.5 ng of pUC18

attaching-effacing genes (A/E) (Table 1) according to published methods (5, 17, using the following settings: 25 F, 2.5 kV, and 200 . The cells were incubated

24). for 1 h in SOC (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10

Phage puri cation. One liter of Hershey medium was inoculated with a bac- mM MgCL2, 20 mM Mg SO4, 20 mM glucose) medium at 37 C and plated on

terial colony, grown to an OD of 0.1, and then infected at a multiplicity of Hershey agar containing 20 g of ampicillin.

The experiments with the Ampr cells were conducted with a total of 21

infection of 5. NaCl was added to the lysate to a nal concentration of 0.5 M and

incubated 1 h at 4 C. After centrifugation at 10,000 rpm (16 min at 4 C) in a animals, i.e., three groups of seven mice per experiment. In each experiment,

three mice received the Ampr cells without phage, three received Ampr cells and

Sorvall RC5B centrifuge, polyethylene glycol 6000 was added to the supernatant

phage in the drinking water, while one mouse received phage orally but no Ampr

to a nal concentration of 10%. The lysate was incubated overnight at 4 C with

gentle stirring. Polyethylene glycol-precipitated phages were collected by cen- cells. Six-week-old C3H male mice were taken for the experiment. The three

trifugation at 14,500 g for 16 min. The resulting pellets were resuspended in 3 experiments differed with respect to addition of oral ampicillin (experiment 1, no

2560 CHIBANI-CHENNOUFI ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1. Susceptibilities of E. coli strains to infection with T4-like phages

Lysisb caused by: Lysisb caused by:

Strain Strain

JS4 JSD.1 JSL.6 JS94.1 JS4 JSD.1 JSL.6 JS94.1

ECOR

EPEC O18:K77 X

1

EPEC O20:K84 X X

2

EPEC O26:K60 X

4 X X

EPEC O55:K59 X X

5

EPEC O86:K61

6

EPEC O111:K58 X

8 X

EPEC O112:K66 X

9

EPEC O119:K69 X

11

EPEC O124:K72 X

12

EPEC O125:K70

13 X

EPEC A/E a X (X)

14 X

EPEC A/E a X

15 X X

EPEC A/E a X X

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24 X X

EPEC A/E a

26 X

EPECa (X) X

28

EPECa

35 X

EPECa (X) X

36 X

EPECa

38 X

39 X

ETEC O6:H16

40 X

ETEC O8:H9 X

41

ETEC O15:H11

42

ETEC O25:H42 X

43

ETEC O78:H12

48 X

ETEC O115:H51

49 (X)

ETEC O20:H11

50

ETEC O27:H7

51

ETEC O128:H18

53

ETEC O63:H

54

ETEC O148:H28 X

55

ETEC O153:H12

56 X

LT /ST ; CFA 1a

ETEC

59

LT /ST ; CS6a

ETEC

60 (X) X

LT /ST ; PCF O166a

ETEC

61 (X)

LT /ST ; CFA1a

ETEC X

62

LT /ST ; CS4; CS6a

ETEC

63 X

LT /ST ; CS1; CS3a

ETEC X (X)

64

LT /ST ; CS5; CS6a

ETEC

71

LT /ST a

ETEC

72

LT /ST a

ETEC

LT /ST a

ETEC X

LT /ST a

ETEC

LT /ST a

ETEC X

a

Isolated from the Dhaka hospital laboratory.

b

X, lysis; (X), partial lysis.

ampicillin; experiment 2, ampicillin was given together with Ampr cells; experi- 2 mice received cells and phage at the same time, while in experiment 3 the mice

ment 3, ampicillin was given rst, followed by Ampr cells). Their drinking water received rst the phage and then the bacteria.

consisted of Vittel supplemented with the four-phage cocktail (106 PFU/ml) and

ampicillin (20 mg/ml) as speci ed in the text. The animals were force fed with

RESULTS

ampicillin-resistant K803 (5 107 CFU) supplemented with 6 mg of ampicillin

as speci ed in the text. Feces were sampled twice a day for the rst 4 days and

Isolation and characterization of the phages. When tested

once a day for the rest of the study. Tenfold dilutions of resuspended feces were

on the E. coli strain K803, stool samples from pediatric diar-

plated on Drigalski agar containing 20 g per ml of ampicillin.

Axenic mice. A total of six 8-week-old C3H axenic male mice from our own rhea patients and environmental water samples in Dhaka, Ban-

animal house breeding colony were allotted to three experiments. Each group

gladesh, and sewage from Switzerland yielded nearly exclu-

consisted of two animals held under sterile conditions in a Makrolon type 2 cage

sively Myoviridae (phages with a contractile tail). The

maintained in the same cage without a ltered lid within a positive pressure

elongated heads measured 110 nm by 75 nm. A collar sepa-

isolator of the animal house. In experiment 1 mice were force-fed with E. coli

rated the head from the tail sheath, which measured about 95

K803 (0.5 ml at a concentration of 108 CFU/ml) using intubation. Daily fecal

samples were investigated for E. coli cell counts on Drigalski plates. One week nm in length and 18 nm in width, with an annular substructure

after colonization, sterile- ltered phage JS94.1 was given continuously at a con-

(Fig. 1A). The tail is terminated by a base plate structure to

centration of 105 PFU/ml in the drinking water followed by daily fecal cell and

which both short tail spikes and 150-nm-long tail bers with a

phage counts. In the next week a second force-feeding was performed with E. coli

central knee joint are attached (Fig. 1A, subpanel b). Phages

ECOR5 followed two weeks later by force-feeding with ECOR56. In experiment

VOL. 48, 2004 BACTERIOLYTIC ACTIVITIES OF E. COLI PHAGES 2561

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FIG. 1. Four T4-like phages used in the mouse experiments. (A) Transmission electron microscopy picture of CsCl density gradient-puri ed

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).

genic E. coli strain collection (Table 1). Most notably, the

with a 40-nm contracted tail sheath were also observed (Fig.

1A, subpanels a and c). In these phages, an internal tail tube combined theoretical host range determined by adding up the

extended beyond the contracted tail sheath. The morphology host ranges of the four individual phages was 19 out of 40

(47%) of the pathogenic E. coli strains. When tested on the

suggests T4-like phages. All phage isolates were tested indi-

vidually in the spot test against a collection of pathogenic E. K803 strain, progeny phage was detected in broth culture in-

coli strains associated with diarrhea. From this screening a fections at about 40 min postinfection and phage titer in-

group of four phages was selected that offered the broadest creased afterwards, sometimes in a biphasic way (Fig. 2B). The

combined theoretical host range. All four phages showed the OD of phage-infected K803 cultures lagged from the beginning

typical morphology of T4-like phages (Fig. 1A); showed, like behind the OD development of the uninfected culture (Fig.

phage T4, a 170-kb genome upon pulsed- eld gel electro- 2A).

The fact that E. coli is also a normal constituent of the gut

phoresis; yielded with gp23- and gp32-speci c primers the di-

agnostic PCR products for T4-like phages (36, 53) (data not ora of humans could present a peculiar problem for phage

therapy of E. coli diarrhea. The four phages constituting the

shown); and showed distinct restriction patterns (Fig. 1B).

These four selected phages were rescreened for their lytic cocktail were therefore also investigated individually for their

potential on pathogenic E. coli strains by a tube lysis test. This lytic potential on nonpathogenic E. coli strains from the ECOR

test is more labor-intensive than the spot test but offers a more collection (30) in the tube lysis test (38). The ECOR collection

is a widely used set of 72 reference Escherichia coli strains

rigorous assessment of the bacterial lysis activities of the test

phages (Table 1). Also in the tube lysis test, the stool phages isolated between 1973 and 1983 from a variety of animal hosts

JS4 and JS94.1 and the environmental water phages JSD.1 and and a variety of geographic locations. In broth culture, JS4,

JSL.6 showed a complementary lytic potential on our patho- JS94.1, JSL.6, and JSD.1 lysed eight, three, three, and ve

2562 CHIBANI-CHENNOUFI ET AL. ANTIMICROB. AGENTS CHEMOTHER.

sample of each mouse was tested over 5 days for the presence

of phage on the K803 indicator cell in the plaque assay. No

phage plaques were detected.

Effect of oral phage on fecal E. coli count in mice. Next we

wanted to determine the threshold for an in vivo lytic effect of

orally applied phages on the intestinal E. coli population in

laboratory mice. To this end the four phages were added as a

cocktail to the drinking water of 10 mice in increasing doses

separated by 3 days of phage-free drinking water. Substantial

variation was seen for the lactose-positive cell count on the

Drigalski plates in all animals, even before phages were added

to the drinking water. This variation was also seen during the

periods of phage feeding to the animals. Using a two-way

analysis of variance with phage dose as a xed factor and

animals as a random factor, we derived the following means

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and standard errors of the means for fecal colony counts on

Drigalski plates: for water only, 106.2 0.04; for 103 phage/ml,

105.9 0.10; for 105 phage/ml, 106.1 0.06; for 107 phage/ml,

105.7 0.06. The effect of the phage dose on the cell count was

highly signi cant (P value 0.0001) but was in absolute terms

very small and thus biologically not signi cant.

In view of the in vitro phage susceptibility of the most prev-

alent lactose-positive fecal colonies on Drigalski agar, the lack

of a bacteriolytic effect of the oral phage on the fecal cell count

was surprising. We considered several hypotheses to explain

this observation. First, under the selective pressure of the

phages the prevalent phage-susceptible strains might have

been replaced by phage-insensitive strains. Second, without

protection (antacid or microencapsulation) phages might not

survive the gastric passage and thus not be available in the

intestine. Third, phages might be present in the gut, but for

FIG. 2. Lysis of E. coli K803 strain by the four T4-like phages in physiological reasons the endogenous intestinal E. coli cell

broth culture. (A) OD development of an uninfected control culture

population resists phage infection.

(K-12) and parallel cultures infected with phages JS94.1, JS4, JSD.1,

The rst hypothesis was addressed by the isolation of 100

and JSL.6. (B) Progeny phage release from the four phage-infected

additional lactose-positive colonies from the feces of two ani-

cultures depicted in panel A. Phage infectivity was measured by plaque

assay. mals during the phage treatment period. The colonies showed

a comparable phage susceptibility pattern before and during

the phage treatment period, leading to the rejection of the

strains, respectively, from 39 ECOR strains included in the test hypothesis of an intestinal outgrowth of phage-resistant E. coli

(Table 1). The combined theoretical host range determined by or other Enterobacteriaceae under the selective pressure of the

adding up the host ranges of the four individual phages was 18 oral phages.

(46%) of the nonpathogenic E. coli strains. Gastrointestinal passage of orally applied phages. The fol-

In the next step, we explored the in vitro lytic activities of the lowing experiments demonstrated that unprotected T4-like

four phages on the endogenous E. coli gut ora from a group phages could survive the gastric passage in conventional adult

of ten conventional adult mice. Over a 10-day observation laboratory mice. These experiments refute the second hypoth-

period, feces were recovered every day for each mouse and esis.

lactose-positive yellow colonies were counted on Drigalski agar To begin, we determined the lowest phage concentration

plates. The fecal cell counts showed an average of 106 CFU/g leading to stable fecal phage excretion. To this end four ani-

(data not shown), which is in agreement with similar results mals received in the drinking water successively the four indi-

obtained by Poulsen et al. (39). Five random colonies were vidual phages added at 10-fold dilution steps. Fecal phage

selected per day for each mouse, and the total of 500 colonies titers decreased with the titer of the phage in the drinking

was tested against the four phages in the spot test to reduce the water in an approximate dose-response pattern (Fig. 3). With

the lowest phage concentration of 103 PFU/ml in the drinking

workload. Between 85 and 100% of the tested colonies were

lysed by phages JS4 and JSD.1. JSL.6 lysed about 80% of the water, only low fecal phage titers over short time periods were

observed, while exposure to 104 PFU/ml resulted in fecal

colonies. More variation was observed with phage JS94.1,

which lysed less than 40% of the isolated murine E. coli gut phage detection nearly over the entire exposure period (Fig.

strains in six animals. When the results were combined, prac- 3).

tically all cells were lysed by one of the E. coli phages, con- In the next experiment, we asked whether the gastrointesti-

rming the attribution of the vast majority of the yellow colo- nal passage differed between the individual phages or between

nies from the feces of conventional mice to E. coli. One fecal individual mice. To answer this question, four mice received

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FIG. 3. Gastrointestinal passage of the orally added phages in conventional mice. Fecal phage titer after oral addition of the speci ed 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 gure. The triangles give the phage titers for the control mice. The periods of phage-free drinking water are indicated by white boxes.

rose from undetectable titers to beyond 1010 PFU/ml (Fig. 4A).

successively each of the four individual phages at a xed con-

centration of 107 PFU/ml in the drinking water. Each phage The 100,000-fold titer increase with respect to the phage con-

addition was followed by a phage-free drinking water period centration in the drinking water documented an active repli-

before the next phage isolate was added to the drinking water. cation of phage JS94.1 in the guts of the experimental animals.

Concomitantly, the fecal E. coli cell count dropped from 108 to

The fecal phage counts were assessed on a daily basis (data not

104 CFU/ml or even lower, documenting a substantial in vivo

shown). Three observations were made in this experiment.

First, no signi cant difference was detected between the indi- bacteriolytic activity of the orally applied phage (Fig. 4A).

vidual animals with respect to gastrointestinal phage passage. Despite this serious drop in host cell density, the very high

Second, some difference in gastrointestinal passage was de- fecal phage titers decreased only slowly over the next four days.

tected between the different phage isolates as documented by Interestingly, over the same time period the fecal cell count

increased from undetectable levels to about 105 CFU/ml,

a 50-fold fecal phage titer difference between mice fed phage

JS94.1 and JSL.6 which achieved the highest and the lowest which is still 1,000-fold lower than the original fecal cell count.

fecal phage titers, respectively (data not shown). Third, in one At days 9 and 11, several dozen colonies were picked. All were

mouse the identity of the fecally excreted phage with the phage sensitive to phage JS94.1 suggesting that some of the cells are

in the drinking water was demonstrated for all four feeding now in gut sites protected from phage.

periods by restriction analysis of the fecally reisolated phage. Two weeks after the colonization with K803, the mice were

Next we asked whether the stool phage isolates could not force-fed the ECOR5 strain that is insensitive to phage JS94.1

in vitro. Within days, the fecal cell count rose to 1010 CFU/ml,

infect their host cells due to the anaerobic atmosphere of the

gut environment. This was not the case: during in vitro growth suggesting successful colonization with the new E. coli strain

in an anaerobic jar, 11 of 32 T4-like stool phage isolates and (Fig. 4A). In parallel with the renewed fecal cell count in-

one of the four test phages (JSL.6) lysed its target cells under crease, the fecal phage titer dropped by 4 logs or more, con-

both anaerobic and aerobic conditions. sistent with the expected replacement of a phage-sensitive by a

Finally, we asked whether phages could be given repetitively phage-insensitive intestinal cell population. Alternatively, the

without interference by an intestinal immune response. Phage phage-sensitive population did not change, but the ability of

JS94.1 was given three times to two mice. Each intervention the phage to infect was diminished under the circumstances

was followed by a 2-week rest period. In each case infectious created. The mice were then force-fed with ECOR56 strain

phage was detected in the stools samples with titers approxi- (sensitive to phage JS94.1) followed by phage JS94.1 in the

mately proportional to concentration of the phage in the drink- drinking water. This operation did not rescue the fecal phage

ing water (data not shown). titer (Fig. 4A), suggesting colonization resistance or host im-

Phage treatment of axenic mice. To test the in vivo lytic mune defenses. At day 42, no phages were detected in four

activities of the isolated stool phages, we inoculated two axenic different rinsed gut segments (duodenum, jejunum, ileum, co-

mice with a single E. coli strain, namely, the indicator cell lon) or in the liver or in mesenteric lymph nodes.

K803, resulting in a cell concentration of 108 CFU/g of feces In the next experiment, two axenic mice were force-fed with

104 K803 cells and received at the same time the four-phage

(Fig. 4A). One week later, the K803-colonized mice were ex-

posed to phage JS94.1 at 105 PFU/ml in the drinking water. cocktail at 106 PFU/ml in the drinking water (data not shown).

One mouse showed an initial high fecal cell count (109 CFU/g

Within a day, the fecal phage titer in the JS94.1-exposed mice

2564 CHIBANI-CHENNOUFI ET AL. ANTIMICROB. AGENTS CHEMOTHER.

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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 speci ed E. coli strains, phage JS94.1, or water at the

speci ed time points; the start day is indicated with an arrow below the abscissa. A black line with squares or a gray line with triangles identi es

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 identi es values from an individual mouse.

of feces), followed by a precipitous drop to 104/g and lower. cation of phages after a reduction of phage titers in the stom-

Another mouse showed a fecal cell count decrease from 106 to ach, but the consequence of a passive transit through the entire

104 CFU/g (data not shown). Both mice showed a fecal titer gastrointestinal tract including the stomach. One week later

the mice were force-fed with 104 K803 cells. Introduction of E.

1,000-fold higher than the drinking water phage titer over the

rst days of the experiment, suggestive of active in vivo phage coli into the gut resulted in a transient 1,000- to 10,000-fold

replication. fecal phage titer increase (Fig. 4B). During the initial phase of

Finally, two axenic mice were rst exposed to the four-phage intestinal phage replication, a low and variable fecal cell num-

cocktail at 106 PFU/ml in the drinking water before receiving ber was observed. This phase was followed by a steady increase

cells. Notably, in the absence of intestinal bacteria, 106 PFU of of bacteria to 109 CFU/g stool over the next days (Fig. 4B), and

phages were also detected per g of stool (Fig. 4B), demonstrat- bacteria remained at this level until 2 weeks later (data not

ing that the fecal phages are not the result of intestinal repli- shown), while the phage titers dropped to low levels.

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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 speci ed 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.

Follow-up of Ampr E. coli cells in conventional mice. The E. coli cells were only transiently observed directly after the

preceding experiments suggested that orally applied phages force-feeding (data not shown). Phage was detected only

lysed only E. coli cells that were recently introduced into the during the phage feeding period with a 1-day time lag for

intestine. To differentiate newly introduced from resident E. appearance and 2 days for disappearance.

coli strains, 108 CFU of K803 cells transformed with plasmid To overcome the colonization resistance of the resident in-

testinal ora against the introduction of new cells, the Ampr-

pUC18 containing an ampicillin resistance marker were

force-fed to three conventional mice. Transient peaks of labeled cells were given together with ampicillin during the

fecal Ampr E. coli cells were detected half a day after the force-feeding. During the rst week ampicillin was also added

to the drinking water (20 g/ml). Under these conditions, 103

force-feeding, but they were lost from the intestine half a

to 105 CFU of the Ampr E. coli cells were detected per g of

day later (data not shown). No spontaneous fecal phage

excretion was seen in these mice or the corresponding con- stool and for at least 5 days maintained after omission of

trol mice of the experiments reported below. Three further ampicillin from the drinking water (Fig. 5A). When similarly

mice received in addition 106 PFU of phage per ml in the treated mice received phage in addition in the drinking water,

drinking water. As in the preceding experiment, fecal Ampr Ampr E. coli cells were detected after day 2 only in two fecal

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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.

samples with low counts, suggesting elimination of Ampr E. at 107 PFU/ml in the drinking water and nally sacri ced. Four

coli from the gut (Fig. 5B) by oral phage. different gut segments (once rinsed with buffer to remove the

In the next experiment, six mice were pretreated with am- gut content), the liver, and the mesenteric lymph nodes were

picillin both by force-feeding and in drinking water before tested for the presence of K803 cell and phages. Neither cells

being fed with Ampr E. coli cells. In this experiment only one ( 10 CFU/ml) nor phages ( 10 PFU/ml) were detected in the

stool sample from three mice receiving phages in the drinking mesenteric lymph nodes or the liver. Four further axenic mice

water showed a low fecal cell count of Ampr cells (Fig. 6B) were sacri ced 4 days after a change to phage-free drinking

compared to 15 stool samples from the three mice receiving water. None of the investigated tissues were associated with

plain water (Fig. 6A). phages (data not shown). Tissue samples from eight phage-

Orally fed T4-like phages remain restricted to the gut. Four treated mice and two control mice were processed for standard

axenic mice were inoculated with K803 cells and then force-fed histology analysis. For the intestinal samples, both longitudina

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