Veterinary Microbiology, 30 ( 1992 ) 203-212 Elsevier Science Publishers B.V., Amsterdam
203
Characteristics and diffusion in the rabbit of a phage for Escherichia coli 0103. Attempts to use this phage for therapy A. Reynaud a, ~, L. Cloastre a, J. Bernard a, H. Laveran b, H.-W. Ackermann c, D. L i c o i s a a n d B. J o l y e aPuy-de-Dfme Departmental Veterinary Laboratory, RN 89, Marmilhat, 63370 Lempdes, France bDepartment of Ultracentrifugation, Faculty of Medicine, University Clermont L 63001 ClermontFerrand, France CF~lix d'Hbrelle Reference Centerfor Bacterial Viruses, Department of Microbiology, Faculty of Medicine, Laval University, Quebec, Qc, G1K 7P4, Canada dlNRA de Tours-Nouzilly, Unitb de Pathologic du Lapin, 37380 Monnaie, France eFaculty of Pharmacy, University Clermont I, 63001 Clermont-Ferrand, France (Accepted 5 July 1991 )
ABSTRACT Reynaud, A., Cloastre, L., Bernard, J., Laveran, H., Ackerman, H.-W., Licois, D. and Joly, B., 1992. Characteristics and diffusion in the rabbit of a phage for Escherichia coli 0103. Attempts to use this phage for therapy. Vet. Microbiol., 30: 203-212. A bacteriophage for Escheriehia coli 0103 was isolated during a study on E. coli diarrhoea in intensive breeding units of rabbits. The phage had an isometric head and a short tail and resembled coliphage N4 (Podoviridae). It had a very narrow host range and seemed to be specific for serogroup 0103, suggesting that it might be used for preliminary identification ofE. coli strains of this serogroup instead of the usual slide agglutination. In view of its possible use as a therapeutic phage, we investigated its dissemination in rabbit organs after oral administration. The phage persisted in the spleen for at least 12 days. However, in vivo studies showed that this phage and a mixture of more virulent phages for E. coli 0103 were ineffective in preventing disease in rabbits inoculated with an enteropathogenic strain of E. coli 0103.
INTRODUCTION
Escherichia coli 0103 causes severe enteritis breeding units in France. The mortality rate 1983, Reynaud et al., 1987; Reynaud et al., source of major economic loss. The present generalized use of antibiotics
in young rabbits in commercial is about 50%, (Renault et al., 1991 ), making E. coli 0103 a has led to the emergence of a
~Author for correspondence.
0378-I 135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
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A. REYNAUD ET AL.
antibiotic-resistant strains and to a renewed interest in phage therapy as an alternative. However, studies on bacteriophage dissemination within animal organisms have been limited to mice and intramuscular inoculation of phages (Smith and Huggins, 1982 ). Generally, the fate of phages in the organism, or their pharmacokinetics, remains practically unknown. We isolated a phage for E. coli 0103 which produced clear lysis on agar, suggesting that it could be used for phage therapy. Because of the general suitability of oral administration for mass treatment by phages, we wanted to know if oral inoculation of rabbits with our phage resulted in persistence of phages in the intestine and their dissemination into internal organs. The purposes of this study were: • to describe morphology, host range and other properties of our phage. • to study phage dissemination within Specific Pathogen Free (SPF) rabbits after intra-stomacal inoculation. • to assay our phage and a mixture of highly virulent phages for therapy of rabbits inoculated with an enteropathogenic strain ofE. coli 0103. MATERIALS AND METHODS
Phage isolation and propagation Phage CF 0103 was isolated in February 1988 from the air of a rabbit breeding unit with endemic diarrhoea caused by E. coli 0103. The phage manifested its presence by multiple plaques. For large-scale production, a flask containing 500 ml of trypticase soy broth (Bio M6rieux, Marcy l'Etoile, France ) was seeded with 5 ml of log-phase E. coli 0103 bacteria and 5 ml of a phage lysate (titer 106 P F U / m l ) . After 3 h at 37°C, the lysate was filtered through membrane filters of 0.45/zm pore size (Sartorius, Palaiseau, France). Titers of 108 P F U / m l were so obtained. The 2 m m diameter plaques were clear, suggesting that the phage was virulent. Buoyant density in CsC1 One liter of lysate was centrifuged for 4 h at 150 000 g (ultracentrifuge Beckman L8-50, rotor SW-28). The sediment was resuspended in phosphate buffered saline (PBS) (pH 7.2), adjusted with CsC1 to a density of 1.45 g/ ml, and centrifuged for 22 h at 180 000 g and 4 ° C in a SW-50 rotor. Fractions (0.5 ml ) were tested for absorbancy at 280 n m using a Beckman DU-64 photometer. Positive fractions were pooled, centrifuged a second time in CsCI, dialyzed against PBS buffer, and titrated on E. coli 0103. Electron microscopy Phages were concentrated and stained with uranyl acetate (2%) or phosphotungstate (2%) as described earlier (Ackermann et al., 1988 ) and exam-
CHARACTERISTICS IN RABBIT OF AN E. COLI PHAGE
205
ined with a Philips EM 300 electron microscope. Magnification was calibrated with catalase crystals (Luftig, 1967 ).
Phage DNA Phages were concentrated using polyethylene glycol (Yamamoto et al., 1970) and centrifugation at 11 000 g for 10 min. The sediment was suspended in 100/d of TE buffer ( 10 mM Tris, 1 mM EDTA, pH 8.0). Proteins were extracted by the phenol-ether method (Maniatis et al., 1978). Phage DNA was precipitated with NaC1 (0.1 M) and ethanol (2.5 vols), sedimented, resuspended in 10 ml TE buffer, and treated with restriction endonuclease EcoR1 as indicated by the manufacturer (GIBCO BRL, Cergy-Pontoise, France). Equal amounts (0.1/tl) of treated and untreated phage DNA were subjected to electrophoresis in a 0.7% agarose gel (type II, Sigma, St. Louis, M0). Bands were stained with 1/tg/ml ethidium bromide (Sigma). Host range Phage suspensions of 105 P F U / m l were assayed by the spot test on bacterial lawns on Mueller-Hinton agar (Bio M6rieux). Plaques were examinated after 24 h at 37°C. Phage CF0103 was tested on (i) the reference strain of E. coli 0103 (strain W2469, serotype, 0:103 : K-:H2 ), obtained from the Statens Serum Institute, Copenhagen, Denmark; and (ii) 163 wild E. coli strains, 97 of them belonging to the 0103 serogroup and 66 being auto-agglutinating or serologically nontypable (Table 1 ). All 163 strains were isolated in the Puyde-D6me Veterinary Laboratory between 1987 and 1989. The presence of 0103 antigen was detected by slide agglutination with 0103 antiserum purchased from INRA Toulouse, France and was confirmed by agglutination with the same 0103 serum after boiling of bacteria for 60 rain (Orskov et al., 1977). A few strains showed auto-agglutination in saline (0.7%) after boiling and were untypable (Table 1 ). (iii) In addition, the phage was tested on a series of E. coli, Salmonella and Shigella dysenteriae strains maintained as phage hosts in the F61ix d'H6relle Reference Center for Bacterial Viruses (Table 1 ). Inoculation of experimental animals Sixty five-week-old specific pathogen free (SPF) rabbits, free of E. coli 0103 (Coudert et al., 1988 ) were obtained from the Station de Pathologie Aviaire et de Parasitologie, INRA, Tours, France. Two days before inoculation with phages, the animals were distributed into cages at a ratio of five per cage and were placed in a controlled-environment room (Coudert et al., 1979). They received ad libitum a laboratory diet free of antibiotics and anti-coccidial agents (UAR 112, 91360 Villemoisson sur Orge, France ). Phage CF0103 was administered by stomach tubes at a concentration of 109 PFU/ml. Each animal received 5 ml of phage suspension.
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Study of phage dissemination Two rabbits were sacrificed on days 1, 2, 4, 7 and 12 after inoculation, respectively. Samples of kidney, liver, spleen, intestinal and caecal content, blood and urine were collected under sterile conditions using gloves and under microbiological safety cabinet (BSB4, Flow Laboratories, Les Ulis, France). Before dissection, animals were disinfected with tincture of iodine. Two grams of each organ sample were placed into 18 ml of sterile physiological saline and then ground with Ultraturax (type 18-10 Janke et Kunkel, D7813 Staufen, F R G ) . Suspensions were first filtered through porous paper and then through membrane filters of 0.45/tl pore size (Sartorius, Palaiseau, France). Samples (2 ml) of ileal and caecal content were added to 9 ml of sterile physiological saline and homogenized with a Vortex mixer (type Polymix, Bioblock Scientific, 67403 Illkirch, France). After 15 min at + 4°C, the mixture was filtered through membrane filters as above. Blood was drawn from the ear vein and heparinized. One ml of sample was added to 9 ml of sterile distilled water and erythrocytes were eliminated by centrifugation at 1300 g for 10 min. Similarly, 1 ml of urine was added to 9 ml of sterile saline. Phage titers were determined for each sample.
Phage therapy Five groups of 10 rabbits each were inoculated as follows: • group 1 with strain GV of pathogenic E. coli 0103 (Reynaud et al., 1991 ). • group 2 with phage CF0103. • group 3 with a mixture, in equal proportions, of five phages different from the phage CF0103. These phages were isolated by the method of Smith and Huggins (1982) from different specimens of sewage from the outskirts of Clermont Ferrand (France). They were assayed because they were lytic for E. coli 0103 and because each of them w a s 1 0 4 times more virulent, both at 37 °C and 39 oC, than phage CF0103. Phage virulence tests were performed according to the m e t h o d of Smith and Huggins ( 1983 ). • group 4 with phage CF0103 and strain GV ofE. coli 0103. • group 5 with a mixture of the above mentioned five phages and E. coli 0103 GV. Phage particles a n d / o r bacteria were administered by stomach tubes at a concentration of 1 0 9 P F U / a n i m a l and 1 0 7 bacteria/animal. Weight gain, mortality and fecal output of E. coli 0103 were recorded daily for 15 days after inoculation according to methods already described (Reynaud et al., 1991 ). Simultaneously, phages for E. coli 0103 were counted daily in faeces. RESULTS
Properties of phage CF0103 Particles had isometric heads of about 60 n m in diameter and short tails of 12 X 8 nm. Phage heads were icosahedral as evidenced by the simultaneous
207
CHARACTERISTICS IN RABBIT OF AN E. COLI PHAGE
presence of capsids with hexagonal and pentagonal outlines. Tails showed 12 nm long spikes. Preparations contained traces of small irregular polyheads (Fig. 1 ). The phage had a buoyant density of 1.5 g/ml and a DNA content of about 40 kb. Digestion with restriction endonuclease EcoR 1 resulted in a single band of about 20 kb, indicating that the DNA was double-stranded in nature and possessed only one EcoR 1 site. Host range of CFOl03 Phage CF0103 lysed the 0103 reference strain and 95 out of 97 strains of this serogroup isolated in the Puy-de-D6me Veterinary Laboratory, but was inactive both against 54 strains which did not belong to this serogroup and against 18 reference strains of the F61ix d'H6relle Reference Center used for propagating type phages (Table 1 ). However, phage CF0103 lysed 7 out of 12 strains of unknown serogroups (Table 1 ). For comparison, coliphage N4
iiiiiiiii~ii
b
d
O
Fig. 1. Morphology of phage CF0103 after staining with uranyl acetate (UA) or phosphotungstare (PT). Final magnification, X297 000; bar indicates 100 nm. a, Phage with hexagonal capsid, UA. Arrows indicate spikes, b, Phage with five-sided capsid, UA. c, PT-stained phage, d, Aspect of particle after UA positive staining ; note the dark staining and shrinkage of phage head as compared to negatively stained particles (a and b). e, Small irregular polyhead, UA.
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A. REYNAUD ET AL.
TABLE l Host range o f phage C F 0103 Bacterial strains Escherichia coli, wild strains ~ 0103 ( N + , H + ) N o n 0103 (N-, H - ) N o n 0103 ( N + , H - ) Serogroup u n k n o w n ( N + , H auto-agglutinating) Reference strains 2 Escherichia coli S a l m o n e l l a spp. Shigella dysenteriae
Total
Lysed
97 35 19 12
95 7
14 3 1
-
q s o l a t e d f r o m rabbit caeca. 2Reference strains for p r o p a g a t i o n o f phage T 1 to T7, 2, N4, PI, ~ X 174 a n d R 17. H, agglutination after boiling o f bacteria for 60 rain. N, slide agglutination. TABLE2 Phage tilers in v a r i o u s organs a n d b o d y fluids after oral a d m i n i s t r a t i o n o f C F 0 1 0 3 (Log10 No. of phage p a r t i c l e s / g o f material ) Site
Kidney Liver Spleen Ileum Caecum Plasma P l a s m a after cell lysis Urine
D a y s after inoculation 1
2
4
7
12
4.7 3.8 3.9 3.6 2.6 ND 2.5
4.3 3.7 6.0 2.8 3.2 ND 2.5
3.3 5.2 3.0 3.5 3.5 ND 4.5 < 1.0
< 1.0 < 1.0 2.8 < 1.0 < 1.0 ND < 1.0 < 1.0
< 1.0 < 1.0 3.8 < 1.0 < 1.0 ND < 1.0 < 1.0
Data are average values for two animals. N D , not d e t e r m i n e d .
was tested on the 18 reference strains of the F61ix d'H6relle Reference Center and lysed seven of them. However, it did not lyse any of 20 strains chose at random among 97 strains of serogroup 0103 (Table 1 ).
Phage dissemination in the rabbit Average numbers of CF0103 particles observed in tissues of two SPF rabbits are shown in Table 2. Phage CF0103 was found in different tissues (kidney, liver, spleen) and intestine (ileum and caecum) on the 1st and 2nd day after inoculation, but not in plasma. The absence of phage particles in plasma
CHARACTERISTICS IN RABBIT OF AN E. COL1 PHAGE
209
is possibly due to adhesion of the phage to red cells or leucocytes. This hypothesis was confirmed by phage counts on the 4th day after inoculation, which revealed 3 X 104 P F U / m l in plasma after lysis of erythrocytes by osmotic shock. In addition, numeration of phage particles showed that (i) Phage titers in ileum and caecum were similar over time. (ii) Phage titers in the kidney were higher than in other organs and phages persisted there longer than in urine. (iii) The spleen was the only organ where the phage could be found at the end of the investigation (Table 2).
Therapeutic assay No mortality was observed in animals inoculated only with phage CF0103 or with the combination of other virulent phages (groups 2 and 3). In these groups, the average weight gain was 35 g/day. E. coli contents of faeces were about 104-105 cells/g of faeces in the first week and rose to 107-108 during the second week. All coliform bacteria isolated during the second week belonged exclusively to the 0103 serogroup and the GV strain as ascertained by agglutination, absence of rhamnose fermentation and antibiotic sensibility. Five colonies were tested with CF0103 and were all lysed by this phage. In animals inoculated with the enteropathogenic GV strain only (group 1 ), rabbits started to die on day 4 after inoculation. Mortality reached a peak between days 5 to 7. At the end of the experiment, 90% of rabbits had died. A severe slowing of weight gain was observed between days 2 and 7 after inoculation and afterwards rabbits continued to lose weight. The number of E. coli 0103 in faeces varied from 107/g of faeces two days after inoculation to 108-109/g later. In rabbits of groups 4 and 5, inoculated with the GV strain and either with phage CF0103 or the phage mixture, the only difference from the previous groups was that the onset of mortality was 7 days after inoculation, but the evolution of weight gain and of the faecal output of E. coli 0103 was strictly similar to the previous groups. In all five groups, the number of coliphages was very high from day 4 after inoculation and reached 109-10 l° particles/g of faeces during the rest of the experiment. These values in control groups 2 and 3 which had not been inoculated with bacteria, could be explained by contamination of animals by the GV strain.
DISCUSSION By its general morphology, phage CF0103 belongs to the Podoviridae family (short-tailed phages ) (Matthews, 1982 ). The presence of conspicuous tail spikes suggests that it is a member of the N4 species of enterobacterial phages (Ackermann and Dubow, 1987; Schito, 1974). However, its attribution to this species remains tentative because of the relatively simple structure of N4type viruses. Interestingly, N4 DNA has not a single EcoR1 cleavage site
210
A. REYNAUD ETAL.
(Zivin et al., 1980). Phage CF0103 differs from N4 in host range and is considered as a new entity which has been included into the F61ix d'H6relle Reference Collection as HER 337. There was a close correlation between agglutination after heating and phage sensitivity. Only two out of 97 heat-agglutinating strains were not lysed by CF0103. In the electron microscope, very numerous CF0103 particles appeared to be adsorbed to the surface of reference strain E. coli 0103 W2469 whereas no such adsorption was observed in two phage-resistant derivatives of this strain (data not shown). This suggested that phage-resistance was due to the absence of receptors. On the other hand, 19 strains which did not agglutinate after boiling, were not lysed. With respect to for identification of 0103 E. coli strains, the relatively high serotype specificity of phage CF0103 suggests than it can be used in veterinary laboratories for screening purposes. Studies on bacteriophage diffusion within animal organs are very few and have been performed only on mice and humans. Smith and Huggins, ( 1982 ) have showed that after intramuscular inoculation of a dose of 3 × 108 viable particles, phages diffused through the whole organism were found in fairly high numbers in blood, spleen and liver. Weber-D~tbrowska et al. (1987), during experimental phage therapy of with septicemia and urinary tract infections, administered phages orally and found that they passed into the bloodstream. Bacteriophages were found in 47 of 56 blood samples collected on the 10th day after administration. Although obtained with rabbits, our results confirm these data. It is clear that orally administered phages can break through the gastrointestinal barrier into the whole organism, are disseminated by blood and reach internal organs, where they are theoretically able to lyse pathogenic bacteria. This observation is very important because oral administration is particularly suited to mass treatment of such animals as rabbits or chickens. In contrast to the relatively long phage persistence in human blood, we found that phage CF0103 was present in blood plasma and most organs for four days only, but persisted in the spleen for more than 12 days. This suggests that they were captured by the reticulo-endothelial system. The rather rapid elimination of phages in non-infected rabbits can be strongly modified in animals infected by E. coli 0103. Indeed, during an examination of dead infected rabbits in intensive breeding units, we have detected bacteriophage CF0103 in caecal contents over two months after a single administration of the phage in drinking water (data not shown). As for phage therapy of rabbits experimentally infected with E. coli 0103, it was surprising that no effect was observed even with highly virulent phages and in spite of a high level of bacteriophages obtained in animals. This contrasted with the known in vitro virulence of these phages, indicating that conditions controlling the gut microflora in animals are complex. For example,
CHARACTERISTICS IN RABBIT OF AN E. COL1 PHAGE
211
diarrhoea occurs in calves, piglets and lambs only on the newborn, while diarrhoea in rabbits develops essentially after weaning. In calves, Smith and Huggins. ( 1983 ) and Smith et al. (1987a) succeeded in controlling diarrhoea induced by enterotoxigenic E. coli (ETEC), characterized by adhesive factors such as K99 antigen. One of their hypotheses was that phagotherapy results in an emergence of resistant mutants that lack these factors, and consequently leads to a greatly reduced virulence. As other E. coli 0103, the GV strain is not an ETEC, but probably an enteropathogenic E. coli (EPEC) (Reynaud et al., 1991 ). The GV strain, like the only phage-resistant E. coli strain of Smith et al. (1987a) has no K antigen. Smith et al., (1987b) also showed that the successful use of phages for controlling bacterial diseases can be affected by factors such as the pH in the alimentary tract, the temperature of animals or the presence of phage neutralizing antibodies. It is clear that the problem of phage treatment of intestinal infections in rabbits needs further study.
REFERENCES Ackermann, H.-W., DuBow, M.S., 1987. Viruses of prokaryotes. Vol. II. Natural groups of bacteriophages. CRC Press, Boca Raton (Florida), 85-100. Ackermann, H.-W., Cartier, C., ~lopek, S. and Vieu, J.-F., 1988. Morphology of Pseudomonas aeruginosa typing phages of the Lindberg set. Ann. Inst. Pasteur/Virol., 39: 389-404. Coudert, P., Licois, D. and Streun, A., 1979. Characterization of Eimeria species. Isolation and study of pathogenicity of a pure strain of Eimeria perforans (Leuckart 1879, Sluiter and Swellengrebel 1912). Z. Parasitenkd., 59: 227-234. Coudert, P., Licois, D. and Besnard, J., 1988. Establishment ofa S.P.F. breeding colony without hysterectomy and hand rearing procedures In: Proc. 4th Congr. World Rabbit Sci. Assoc., October 10-14. Budapest, pp. 137-148. Luftig, R., 1967. An accurate measurement of the catalase crystal period and its use as an internal marker of electron microscopy. J. Ultrastruct. Res., 20: 91-102. Maniatis, T., Hardison, R.C., Lacy, E., Lauer, J., O'Connell, C., Quon, D., Sim, G.K. and Efstratiadis, A., 1978. The isolation of structural genes from libraries ofeucaryotic DNA. Cell, 15: 687-701. Matthews, R.E.F., 1982. Classification and nomenclature of viruses. Fourth report of the International Committee on Taxonomy of Viruses. Intervirology, 17: 1-199. Orskov, I., Orskov, F., Jann, B., and Jann, K., 1977. Serology, chemistry, and genetics of O and K antigens ofEscherichia coli. Bacteriol. Rev., 41:667-710. Renault, L., Roux, J., Le Bourhis, E., Coudert, P., Licois, D. and Guillot, J.F., 1983. Description d'un s6rogroupe 0103 d'Escherichia coli ent6ropathog~ne chez le lapin au sevrage. Bull. Acad. Vet. France, 56: 387-400. Reynaud, A., Federighi, M., Joly, B., Hervouet, P. and Camguilhem, R., 1987. Sp6cificit6, antibiosensibilit6 et virulence des souches d'Escherichia coli isol6s dans le caecum des lapins. Cuni-Sci., 4: 1-9. Reynaud, A., Federighi, M., Licois, D., Guillot, J.F. and Joly, B., R plasmid coding for colonization of the rabbit intestinal tract in Escherichia coli 0103. Infect. Immun., 59:1888-1892. Schito, G.C., 1974. I1 modello del batteriofago N4. Ig. Mod., 67:871-900.
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Smith, H.W. and Huggins, M.B., 1982. Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics. J. Gen. Microbiol., 128: 307-318. Smith, H.W. and Huggins, M.B., 1983. Effectiveness of phages in treating experimental Escherichia coli diarrhoea in calves, piglets and lambs. J. Gen. Microbiol., 129: 2659-2675. Smith, H.W., Huggins, M.B. and Shaw, K.M., 1987a. The control of experimental Escherichia coli diarrhoea in calves by means of bacteriophages. J. Gen. Microbiol., 133:1111-1126. Smith, H.W., Huggins, M.B. and Shaw, K.M., 1987b. Factors influencing the survival and multiplication of bacteriophages in calves and in their environment. J. Gen. Microbiol., 133: 1127-1135. Weber-Da~browska, B., D~browski, M., and Slopek, S., 1987. Studies on bacteriophage penetration in patients subjected to phage therapy. Arch. Immunol. Ther. Exp., 35: 563-568. Yamamoto, K.R., Alberts, B.M., Benzinger, R., Lawhorne, L. and Treiber, G., 1970. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology, 40: 734-744. Zivin, R., Malone, C. and Rothman-Denes, L.B., 1980. Physical map of coliphage N4 DNA. Virology, 104: 205-218.
203
Characteristics and diffusion in the rabbit of a phage for Escherichia coli 0103. Attempts to use this phage for therapy A. Reynaud a, ~, L. Cloastre a, J. Bernard a, H. Laveran b, H.-W. Ackermann c, D. L i c o i s a a n d B. J o l y e aPuy-de-Dfme Departmental Veterinary Laboratory, RN 89, Marmilhat, 63370 Lempdes, France bDepartment of Ultracentrifugation, Faculty of Medicine, University Clermont L 63001 ClermontFerrand, France CF~lix d'Hbrelle Reference Centerfor Bacterial Viruses, Department of Microbiology, Faculty of Medicine, Laval University, Quebec, Qc, G1K 7P4, Canada dlNRA de Tours-Nouzilly, Unitb de Pathologic du Lapin, 37380 Monnaie, France eFaculty of Pharmacy, University Clermont I, 63001 Clermont-Ferrand, France (Accepted 5 July 1991 )
ABSTRACT Reynaud, A., Cloastre, L., Bernard, J., Laveran, H., Ackerman, H.-W., Licois, D. and Joly, B., 1992. Characteristics and diffusion in the rabbit of a phage for Escherichia coli 0103. Attempts to use this phage for therapy. Vet. Microbiol., 30: 203-212. A bacteriophage for Escheriehia coli 0103 was isolated during a study on E. coli diarrhoea in intensive breeding units of rabbits. The phage had an isometric head and a short tail and resembled coliphage N4 (Podoviridae). It had a very narrow host range and seemed to be specific for serogroup 0103, suggesting that it might be used for preliminary identification ofE. coli strains of this serogroup instead of the usual slide agglutination. In view of its possible use as a therapeutic phage, we investigated its dissemination in rabbit organs after oral administration. The phage persisted in the spleen for at least 12 days. However, in vivo studies showed that this phage and a mixture of more virulent phages for E. coli 0103 were ineffective in preventing disease in rabbits inoculated with an enteropathogenic strain of E. coli 0103.
INTRODUCTION
Escherichia coli 0103 causes severe enteritis breeding units in France. The mortality rate 1983, Reynaud et al., 1987; Reynaud et al., source of major economic loss. The present generalized use of antibiotics
in young rabbits in commercial is about 50%, (Renault et al., 1991 ), making E. coli 0103 a has led to the emergence of a
~Author for correspondence.
0378-I 135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
204
A. REYNAUD ET AL.
antibiotic-resistant strains and to a renewed interest in phage therapy as an alternative. However, studies on bacteriophage dissemination within animal organisms have been limited to mice and intramuscular inoculation of phages (Smith and Huggins, 1982 ). Generally, the fate of phages in the organism, or their pharmacokinetics, remains practically unknown. We isolated a phage for E. coli 0103 which produced clear lysis on agar, suggesting that it could be used for phage therapy. Because of the general suitability of oral administration for mass treatment by phages, we wanted to know if oral inoculation of rabbits with our phage resulted in persistence of phages in the intestine and their dissemination into internal organs. The purposes of this study were: • to describe morphology, host range and other properties of our phage. • to study phage dissemination within Specific Pathogen Free (SPF) rabbits after intra-stomacal inoculation. • to assay our phage and a mixture of highly virulent phages for therapy of rabbits inoculated with an enteropathogenic strain ofE. coli 0103. MATERIALS AND METHODS
Phage isolation and propagation Phage CF 0103 was isolated in February 1988 from the air of a rabbit breeding unit with endemic diarrhoea caused by E. coli 0103. The phage manifested its presence by multiple plaques. For large-scale production, a flask containing 500 ml of trypticase soy broth (Bio M6rieux, Marcy l'Etoile, France ) was seeded with 5 ml of log-phase E. coli 0103 bacteria and 5 ml of a phage lysate (titer 106 P F U / m l ) . After 3 h at 37°C, the lysate was filtered through membrane filters of 0.45/zm pore size (Sartorius, Palaiseau, France). Titers of 108 P F U / m l were so obtained. The 2 m m diameter plaques were clear, suggesting that the phage was virulent. Buoyant density in CsC1 One liter of lysate was centrifuged for 4 h at 150 000 g (ultracentrifuge Beckman L8-50, rotor SW-28). The sediment was resuspended in phosphate buffered saline (PBS) (pH 7.2), adjusted with CsC1 to a density of 1.45 g/ ml, and centrifuged for 22 h at 180 000 g and 4 ° C in a SW-50 rotor. Fractions (0.5 ml ) were tested for absorbancy at 280 n m using a Beckman DU-64 photometer. Positive fractions were pooled, centrifuged a second time in CsCI, dialyzed against PBS buffer, and titrated on E. coli 0103. Electron microscopy Phages were concentrated and stained with uranyl acetate (2%) or phosphotungstate (2%) as described earlier (Ackermann et al., 1988 ) and exam-
CHARACTERISTICS IN RABBIT OF AN E. COLI PHAGE
205
ined with a Philips EM 300 electron microscope. Magnification was calibrated with catalase crystals (Luftig, 1967 ).
Phage DNA Phages were concentrated using polyethylene glycol (Yamamoto et al., 1970) and centrifugation at 11 000 g for 10 min. The sediment was suspended in 100/d of TE buffer ( 10 mM Tris, 1 mM EDTA, pH 8.0). Proteins were extracted by the phenol-ether method (Maniatis et al., 1978). Phage DNA was precipitated with NaC1 (0.1 M) and ethanol (2.5 vols), sedimented, resuspended in 10 ml TE buffer, and treated with restriction endonuclease EcoR1 as indicated by the manufacturer (GIBCO BRL, Cergy-Pontoise, France). Equal amounts (0.1/tl) of treated and untreated phage DNA were subjected to electrophoresis in a 0.7% agarose gel (type II, Sigma, St. Louis, M0). Bands were stained with 1/tg/ml ethidium bromide (Sigma). Host range Phage suspensions of 105 P F U / m l were assayed by the spot test on bacterial lawns on Mueller-Hinton agar (Bio M6rieux). Plaques were examinated after 24 h at 37°C. Phage CF0103 was tested on (i) the reference strain of E. coli 0103 (strain W2469, serotype, 0:103 : K-:H2 ), obtained from the Statens Serum Institute, Copenhagen, Denmark; and (ii) 163 wild E. coli strains, 97 of them belonging to the 0103 serogroup and 66 being auto-agglutinating or serologically nontypable (Table 1 ). All 163 strains were isolated in the Puyde-D6me Veterinary Laboratory between 1987 and 1989. The presence of 0103 antigen was detected by slide agglutination with 0103 antiserum purchased from INRA Toulouse, France and was confirmed by agglutination with the same 0103 serum after boiling of bacteria for 60 rain (Orskov et al., 1977). A few strains showed auto-agglutination in saline (0.7%) after boiling and were untypable (Table 1 ). (iii) In addition, the phage was tested on a series of E. coli, Salmonella and Shigella dysenteriae strains maintained as phage hosts in the F61ix d'H6relle Reference Center for Bacterial Viruses (Table 1 ). Inoculation of experimental animals Sixty five-week-old specific pathogen free (SPF) rabbits, free of E. coli 0103 (Coudert et al., 1988 ) were obtained from the Station de Pathologie Aviaire et de Parasitologie, INRA, Tours, France. Two days before inoculation with phages, the animals were distributed into cages at a ratio of five per cage and were placed in a controlled-environment room (Coudert et al., 1979). They received ad libitum a laboratory diet free of antibiotics and anti-coccidial agents (UAR 112, 91360 Villemoisson sur Orge, France ). Phage CF0103 was administered by stomach tubes at a concentration of 109 PFU/ml. Each animal received 5 ml of phage suspension.
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Study of phage dissemination Two rabbits were sacrificed on days 1, 2, 4, 7 and 12 after inoculation, respectively. Samples of kidney, liver, spleen, intestinal and caecal content, blood and urine were collected under sterile conditions using gloves and under microbiological safety cabinet (BSB4, Flow Laboratories, Les Ulis, France). Before dissection, animals were disinfected with tincture of iodine. Two grams of each organ sample were placed into 18 ml of sterile physiological saline and then ground with Ultraturax (type 18-10 Janke et Kunkel, D7813 Staufen, F R G ) . Suspensions were first filtered through porous paper and then through membrane filters of 0.45/tl pore size (Sartorius, Palaiseau, France). Samples (2 ml) of ileal and caecal content were added to 9 ml of sterile physiological saline and homogenized with a Vortex mixer (type Polymix, Bioblock Scientific, 67403 Illkirch, France). After 15 min at + 4°C, the mixture was filtered through membrane filters as above. Blood was drawn from the ear vein and heparinized. One ml of sample was added to 9 ml of sterile distilled water and erythrocytes were eliminated by centrifugation at 1300 g for 10 min. Similarly, 1 ml of urine was added to 9 ml of sterile saline. Phage titers were determined for each sample.
Phage therapy Five groups of 10 rabbits each were inoculated as follows: • group 1 with strain GV of pathogenic E. coli 0103 (Reynaud et al., 1991 ). • group 2 with phage CF0103. • group 3 with a mixture, in equal proportions, of five phages different from the phage CF0103. These phages were isolated by the method of Smith and Huggins (1982) from different specimens of sewage from the outskirts of Clermont Ferrand (France). They were assayed because they were lytic for E. coli 0103 and because each of them w a s 1 0 4 times more virulent, both at 37 °C and 39 oC, than phage CF0103. Phage virulence tests were performed according to the m e t h o d of Smith and Huggins ( 1983 ). • group 4 with phage CF0103 and strain GV ofE. coli 0103. • group 5 with a mixture of the above mentioned five phages and E. coli 0103 GV. Phage particles a n d / o r bacteria were administered by stomach tubes at a concentration of 1 0 9 P F U / a n i m a l and 1 0 7 bacteria/animal. Weight gain, mortality and fecal output of E. coli 0103 were recorded daily for 15 days after inoculation according to methods already described (Reynaud et al., 1991 ). Simultaneously, phages for E. coli 0103 were counted daily in faeces. RESULTS
Properties of phage CF0103 Particles had isometric heads of about 60 n m in diameter and short tails of 12 X 8 nm. Phage heads were icosahedral as evidenced by the simultaneous
207
CHARACTERISTICS IN RABBIT OF AN E. COLI PHAGE
presence of capsids with hexagonal and pentagonal outlines. Tails showed 12 nm long spikes. Preparations contained traces of small irregular polyheads (Fig. 1 ). The phage had a buoyant density of 1.5 g/ml and a DNA content of about 40 kb. Digestion with restriction endonuclease EcoR 1 resulted in a single band of about 20 kb, indicating that the DNA was double-stranded in nature and possessed only one EcoR 1 site. Host range of CFOl03 Phage CF0103 lysed the 0103 reference strain and 95 out of 97 strains of this serogroup isolated in the Puy-de-D6me Veterinary Laboratory, but was inactive both against 54 strains which did not belong to this serogroup and against 18 reference strains of the F61ix d'H6relle Reference Center used for propagating type phages (Table 1 ). However, phage CF0103 lysed 7 out of 12 strains of unknown serogroups (Table 1 ). For comparison, coliphage N4
iiiiiiiii~ii
b
d
O
Fig. 1. Morphology of phage CF0103 after staining with uranyl acetate (UA) or phosphotungstare (PT). Final magnification, X297 000; bar indicates 100 nm. a, Phage with hexagonal capsid, UA. Arrows indicate spikes, b, Phage with five-sided capsid, UA. c, PT-stained phage, d, Aspect of particle after UA positive staining ; note the dark staining and shrinkage of phage head as compared to negatively stained particles (a and b). e, Small irregular polyhead, UA.
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TABLE l Host range o f phage C F 0103 Bacterial strains Escherichia coli, wild strains ~ 0103 ( N + , H + ) N o n 0103 (N-, H - ) N o n 0103 ( N + , H - ) Serogroup u n k n o w n ( N + , H auto-agglutinating) Reference strains 2 Escherichia coli S a l m o n e l l a spp. Shigella dysenteriae
Total
Lysed
97 35 19 12
95 7
14 3 1
-
q s o l a t e d f r o m rabbit caeca. 2Reference strains for p r o p a g a t i o n o f phage T 1 to T7, 2, N4, PI, ~ X 174 a n d R 17. H, agglutination after boiling o f bacteria for 60 rain. N, slide agglutination. TABLE2 Phage tilers in v a r i o u s organs a n d b o d y fluids after oral a d m i n i s t r a t i o n o f C F 0 1 0 3 (Log10 No. of phage p a r t i c l e s / g o f material ) Site
Kidney Liver Spleen Ileum Caecum Plasma P l a s m a after cell lysis Urine
D a y s after inoculation 1
2
4
7
12
4.7 3.8 3.9 3.6 2.6 ND 2.5
4.3 3.7 6.0 2.8 3.2 ND 2.5
3.3 5.2 3.0 3.5 3.5 ND 4.5 < 1.0
< 1.0 < 1.0 2.8 < 1.0 < 1.0 ND < 1.0 < 1.0
< 1.0 < 1.0 3.8 < 1.0 < 1.0 ND < 1.0 < 1.0
Data are average values for two animals. N D , not d e t e r m i n e d .
was tested on the 18 reference strains of the F61ix d'H6relle Reference Center and lysed seven of them. However, it did not lyse any of 20 strains chose at random among 97 strains of serogroup 0103 (Table 1 ).
Phage dissemination in the rabbit Average numbers of CF0103 particles observed in tissues of two SPF rabbits are shown in Table 2. Phage CF0103 was found in different tissues (kidney, liver, spleen) and intestine (ileum and caecum) on the 1st and 2nd day after inoculation, but not in plasma. The absence of phage particles in plasma
CHARACTERISTICS IN RABBIT OF AN E. COL1 PHAGE
209
is possibly due to adhesion of the phage to red cells or leucocytes. This hypothesis was confirmed by phage counts on the 4th day after inoculation, which revealed 3 X 104 P F U / m l in plasma after lysis of erythrocytes by osmotic shock. In addition, numeration of phage particles showed that (i) Phage titers in ileum and caecum were similar over time. (ii) Phage titers in the kidney were higher than in other organs and phages persisted there longer than in urine. (iii) The spleen was the only organ where the phage could be found at the end of the investigation (Table 2).
Therapeutic assay No mortality was observed in animals inoculated only with phage CF0103 or with the combination of other virulent phages (groups 2 and 3). In these groups, the average weight gain was 35 g/day. E. coli contents of faeces were about 104-105 cells/g of faeces in the first week and rose to 107-108 during the second week. All coliform bacteria isolated during the second week belonged exclusively to the 0103 serogroup and the GV strain as ascertained by agglutination, absence of rhamnose fermentation and antibiotic sensibility. Five colonies were tested with CF0103 and were all lysed by this phage. In animals inoculated with the enteropathogenic GV strain only (group 1 ), rabbits started to die on day 4 after inoculation. Mortality reached a peak between days 5 to 7. At the end of the experiment, 90% of rabbits had died. A severe slowing of weight gain was observed between days 2 and 7 after inoculation and afterwards rabbits continued to lose weight. The number of E. coli 0103 in faeces varied from 107/g of faeces two days after inoculation to 108-109/g later. In rabbits of groups 4 and 5, inoculated with the GV strain and either with phage CF0103 or the phage mixture, the only difference from the previous groups was that the onset of mortality was 7 days after inoculation, but the evolution of weight gain and of the faecal output of E. coli 0103 was strictly similar to the previous groups. In all five groups, the number of coliphages was very high from day 4 after inoculation and reached 109-10 l° particles/g of faeces during the rest of the experiment. These values in control groups 2 and 3 which had not been inoculated with bacteria, could be explained by contamination of animals by the GV strain.
DISCUSSION By its general morphology, phage CF0103 belongs to the Podoviridae family (short-tailed phages ) (Matthews, 1982 ). The presence of conspicuous tail spikes suggests that it is a member of the N4 species of enterobacterial phages (Ackermann and Dubow, 1987; Schito, 1974). However, its attribution to this species remains tentative because of the relatively simple structure of N4type viruses. Interestingly, N4 DNA has not a single EcoR1 cleavage site
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A. REYNAUD ETAL.
(Zivin et al., 1980). Phage CF0103 differs from N4 in host range and is considered as a new entity which has been included into the F61ix d'H6relle Reference Collection as HER 337. There was a close correlation between agglutination after heating and phage sensitivity. Only two out of 97 heat-agglutinating strains were not lysed by CF0103. In the electron microscope, very numerous CF0103 particles appeared to be adsorbed to the surface of reference strain E. coli 0103 W2469 whereas no such adsorption was observed in two phage-resistant derivatives of this strain (data not shown). This suggested that phage-resistance was due to the absence of receptors. On the other hand, 19 strains which did not agglutinate after boiling, were not lysed. With respect to for identification of 0103 E. coli strains, the relatively high serotype specificity of phage CF0103 suggests than it can be used in veterinary laboratories for screening purposes. Studies on bacteriophage diffusion within animal organs are very few and have been performed only on mice and humans. Smith and Huggins, ( 1982 ) have showed that after intramuscular inoculation of a dose of 3 × 108 viable particles, phages diffused through the whole organism were found in fairly high numbers in blood, spleen and liver. Weber-D~tbrowska et al. (1987), during experimental phage therapy of with septicemia and urinary tract infections, administered phages orally and found that they passed into the bloodstream. Bacteriophages were found in 47 of 56 blood samples collected on the 10th day after administration. Although obtained with rabbits, our results confirm these data. It is clear that orally administered phages can break through the gastrointestinal barrier into the whole organism, are disseminated by blood and reach internal organs, where they are theoretically able to lyse pathogenic bacteria. This observation is very important because oral administration is particularly suited to mass treatment of such animals as rabbits or chickens. In contrast to the relatively long phage persistence in human blood, we found that phage CF0103 was present in blood plasma and most organs for four days only, but persisted in the spleen for more than 12 days. This suggests that they were captured by the reticulo-endothelial system. The rather rapid elimination of phages in non-infected rabbits can be strongly modified in animals infected by E. coli 0103. Indeed, during an examination of dead infected rabbits in intensive breeding units, we have detected bacteriophage CF0103 in caecal contents over two months after a single administration of the phage in drinking water (data not shown). As for phage therapy of rabbits experimentally infected with E. coli 0103, it was surprising that no effect was observed even with highly virulent phages and in spite of a high level of bacteriophages obtained in animals. This contrasted with the known in vitro virulence of these phages, indicating that conditions controlling the gut microflora in animals are complex. For example,
CHARACTERISTICS IN RABBIT OF AN E. COL1 PHAGE
211
diarrhoea occurs in calves, piglets and lambs only on the newborn, while diarrhoea in rabbits develops essentially after weaning. In calves, Smith and Huggins. ( 1983 ) and Smith et al. (1987a) succeeded in controlling diarrhoea induced by enterotoxigenic E. coli (ETEC), characterized by adhesive factors such as K99 antigen. One of their hypotheses was that phagotherapy results in an emergence of resistant mutants that lack these factors, and consequently leads to a greatly reduced virulence. As other E. coli 0103, the GV strain is not an ETEC, but probably an enteropathogenic E. coli (EPEC) (Reynaud et al., 1991 ). The GV strain, like the only phage-resistant E. coli strain of Smith et al. (1987a) has no K antigen. Smith et al., (1987b) also showed that the successful use of phages for controlling bacterial diseases can be affected by factors such as the pH in the alimentary tract, the temperature of animals or the presence of phage neutralizing antibodies. It is clear that the problem of phage treatment of intestinal infections in rabbits needs further study.
REFERENCES Ackermann, H.-W., DuBow, M.S., 1987. Viruses of prokaryotes. Vol. II. Natural groups of bacteriophages. CRC Press, Boca Raton (Florida), 85-100. Ackermann, H.-W., Cartier, C., ~lopek, S. and Vieu, J.-F., 1988. Morphology of Pseudomonas aeruginosa typing phages of the Lindberg set. Ann. Inst. Pasteur/Virol., 39: 389-404. Coudert, P., Licois, D. and Streun, A., 1979. Characterization of Eimeria species. Isolation and study of pathogenicity of a pure strain of Eimeria perforans (Leuckart 1879, Sluiter and Swellengrebel 1912). Z. Parasitenkd., 59: 227-234. Coudert, P., Licois, D. and Besnard, J., 1988. Establishment ofa S.P.F. breeding colony without hysterectomy and hand rearing procedures In: Proc. 4th Congr. World Rabbit Sci. Assoc., October 10-14. Budapest, pp. 137-148. Luftig, R., 1967. An accurate measurement of the catalase crystal period and its use as an internal marker of electron microscopy. J. Ultrastruct. Res., 20: 91-102. Maniatis, T., Hardison, R.C., Lacy, E., Lauer, J., O'Connell, C., Quon, D., Sim, G.K. and Efstratiadis, A., 1978. The isolation of structural genes from libraries ofeucaryotic DNA. Cell, 15: 687-701. Matthews, R.E.F., 1982. Classification and nomenclature of viruses. Fourth report of the International Committee on Taxonomy of Viruses. Intervirology, 17: 1-199. Orskov, I., Orskov, F., Jann, B., and Jann, K., 1977. Serology, chemistry, and genetics of O and K antigens ofEscherichia coli. Bacteriol. Rev., 41:667-710. Renault, L., Roux, J., Le Bourhis, E., Coudert, P., Licois, D. and Guillot, J.F., 1983. Description d'un s6rogroupe 0103 d'Escherichia coli ent6ropathog~ne chez le lapin au sevrage. Bull. Acad. Vet. France, 56: 387-400. Reynaud, A., Federighi, M., Joly, B., Hervouet, P. and Camguilhem, R., 1987. Sp6cificit6, antibiosensibilit6 et virulence des souches d'Escherichia coli isol6s dans le caecum des lapins. Cuni-Sci., 4: 1-9. Reynaud, A., Federighi, M., Licois, D., Guillot, J.F. and Joly, B., R plasmid coding for colonization of the rabbit intestinal tract in Escherichia coli 0103. Infect. Immun., 59:1888-1892. Schito, G.C., 1974. I1 modello del batteriofago N4. Ig. Mod., 67:871-900.
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Smith, H.W. and Huggins, M.B., 1982. Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics. J. Gen. Microbiol., 128: 307-318. Smith, H.W. and Huggins, M.B., 1983. Effectiveness of phages in treating experimental Escherichia coli diarrhoea in calves, piglets and lambs. J. Gen. Microbiol., 129: 2659-2675. Smith, H.W., Huggins, M.B. and Shaw, K.M., 1987a. The control of experimental Escherichia coli diarrhoea in calves by means of bacteriophages. J. Gen. Microbiol., 133:1111-1126. Smith, H.W., Huggins, M.B. and Shaw, K.M., 1987b. Factors influencing the survival and multiplication of bacteriophages in calves and in their environment. J. Gen. Microbiol., 133: 1127-1135. Weber-Da~browska, B., D~browski, M., and Slopek, S., 1987. Studies on bacteriophage penetration in patients subjected to phage therapy. Arch. Immunol. Ther. Exp., 35: 563-568. Yamamoto, K.R., Alberts, B.M., Benzinger, R., Lawhorne, L. and Treiber, G., 1970. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology, 40: 734-744. Zivin, R., Malone, C. and Rothman-Denes, L.B., 1980. Physical map of coliphage N4 DNA. Virology, 104: 205-218.
