Effectiveness of phages in treating experimental Escherichia coli diarrhoea in calves, piglets and lambs

A mixture of two phages, B44/1 and B44/2, protected calves against a potentially lethal oral infection with an O9:K30,99 enteropathogenic strain of Escherichia coli, called B44, when given before, but not after, the onset of diarrhoea; a mixture in which phage B44/3 was replaced by phage B44/3 was effective after the onset of diarrhoea. Calves that responded to phage treatment had much lower numbers of E. coli B44 in their alimentary tract than untreated calves. Usually, high numbers of phage B44/1 and rather lower numbers of phage B44/2 or B44/3 were present in the alimentary tract of these animals. At death, most calves that had not responded to treatment with phages B44/1 and B44/2 had high numbers of mutants of E. coli B44 resistant to phage B44/1 in their small intestine. Phage-treated calves that survived E. coli infection continued to excrete phage in their faeces, at least until the numbers of E. coli B44 also excreted were low. The phages survived longer than E. coli B44 in faecal samples taken from phage-treated calves and exposed to the atmosphere in an unheated animal house. Calves inoculated orally with faecal samples from phage-treated calves that contained sufficient E. coli B44 to cause a lethal infection remained healthy. A mixture of two phages, P433/1 and P433/2, and phage P433/1 alone cured diarrhoea in piglets caused by an O20:K101,987P strain of E. coli called P433. The numbers of the infecting bacteria and phages in the alimentary tract of the piglets resembled those in the calves. Another phage given to lambs 8 h after they were infected with an O8:K85,99 enteropathogenic strain of E. coli, called S13, reduced the numbers of these organisms in the alimentary tract and had an ameliorating effect on the course of the disease. No phage-resistant mutants of E. coli S13 were isolated from the lambs. The only mutants of E. coli B44 and P433 that emerged in the calves and piglets were K30- or K101- and resistant to phage B44/1 or P433/1 respectively; those tested were much less virulent than their parent strains.     

Restriction of lambda trp bacteriophages by Escherichia coli K

trp-transducing derivatives of phage λ have been used to study Escherichia coli K specific restriction in vivo. The expression of the trp genes from unmodified phages during infection of a rec⁺, restricting host is eliminated by restriction. In a K-restricting recB,C host, where degradation of restricted phage DNA is prevented, expression of the trp genes is little affected by the presence of a single unmodified, K-restriction recognition site, even when that site is within the trpE gene. RI restriction, in contrast to K restriction, prevents trp gene expression in a recB,C host when the restriction target is between the trp genes and the relevant promoter. The presence of two K-restriction recognition sites in a λtrp phage can have a marked effect on trp gene expression. This effect can be interpreted as the result of preferential breakage between the two restriction recognition sites. We conclude that K restriction does not break susceptible DNA at, or even preferentially near, a restriction recognition sequence.     

Degradation studies on Escherichia coli capsular polysaccharides by bacteriophages

The serologically and structurally related Escherichia coli capsular polysaccharides (K antigens) K13, K20, and K23 were found to be depolymerized by the bacteriophages ΦK13 and ΦK20 to almost similar oligomer profiles as shown by polyacrylamide gel electrophoresis. The phage-polysaccharide interactions were followed by an increase of reducing 2-keto-3-deoxyoctulosonic acid due to a phage-associated glycanase that catalyzed the hydrolytic cleavage of common β-ketopyranosidic 2-keto-3-deoxyoctulosonic acid linkages. The related E. coli K antigens K18, K22, and K100 as well as the Haemophilus influenzae type b capsular polysaccharide were degraded by bacteriophage ΦK100 with different efficacy. It is suggested that ΦK100 enzymatically cleaves ribitol-5-phosphate bonds as the only structural feature present in all the polysaccharides investigated.     

Characterization of Escherichia coli donor-specific RNA-containing bacteriophages

The biological properties of 9 clones of Ri bacteriophages isolated from sewage water in 1981 were studied. On the basis of the activity of Ri phages with respect to E. coli donor-specific strains K12, the type of negative colonies, the ultrastructure of the virion and its sizes, adsorption on the pili of host cells, the latent period, the amount of harvest obtained from one infected cell, the clones under study were classified with small spherical RNA-bacteriophages. The neutralization of Ri phages with antiphage sera to standard phages f2 and fr made it possible to classify them with the first serological group and to divide them into 3 subgroups.     

Isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and lambs with diarrhoea in India

AIMS: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in calves and lambs with diarrhoea in India. METHODS AND RESULTS: Faecal samples originating from 391 calves and 101 lambs which had diarrhoea were screened for presence of E. coli. A total number of 309 (249 bovine and 60 ovine) E. coli strains were isolated. A total of 113 bovine and 15 ovine strains were subjected to multiplex polymerase chain reaction (m-PCR) for detection of stx1, stx2, eaeA and EHEC hlyA genes. STEC and EPEC belonging to different serogpoups were detected in 9.73% of calves studied. Six per cent and 26.66% of lambs studied were carrying STEC and EPEC, respectively. Majority of the STEC serogroups isolated in this study did not belong to those which have been identified earlier to be associated mainly with diarrhoea and enteritis in cattle and sheep outside India. The most frequent serogroup among bovine and ovine EPEC was O26 (40%). One of the most important STEC serogroup O157, known for certain life-threatening infections in humans, was isolated from both bovine and ovine faecal samples. CONCLUSIONS: A high percentage of STEC and EPEC belonging to different serogroups are prevalent in calves and lambs with diarrhoea in India and could be the cause of disease in them. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reports, for the first time, the isolation and characterization of STEC and EPEC serogroups associated with diarrhoea in calves and lambs in India. Many STEC and EPEC strains belonged to serogoups known for certain life-threatening diseases in humans.     

Pathogenic effect of enterotoxigenic Escherichia coli and Escherichia coli causing infantile diarrhoea.

Macroscopic, light and electron microscopic alterations in ligated rabbit intestinal loops challenged with five standard enterotoxigenic Escherichia coli (ETEC) and twenty-three enteropathogenic E. coli (EEC-I) strains, freshly isolated from infantile enteritis cases, were investigated. Only two O26 : K60 : H11 strains produced enterotoxin. Their living cultures, sterile filtrates of the fluid medium and ultrasonic lysates of the bacteria resulted in pronounced hypersecretion of the intestinal epithelium followed by fluid accumulation and loop dilatation. These two E. coli strains, similarly as the other loop-negative EEC-I strains, were able to penetrate into the intestinal epithelium. In contrast to the standard ETEC strains, the EEC-I bacteria, adhering to the brush border, intruded into the microvilli, multiplied on the outer epithelial cell membrane making close contact with it and, causing, shedding of microvilli, penetrated into enterocytes becoming enclosed in membrane-bound phagosome-like vacuoles, appeared in the lamina propria and elicited mild focal polymorphonuclear infiltration.     

The ecology of Escherichia coli in calves reared as dairy-cow replacements

A continual turn-over in the strains forming the majority of faecal Escherichia coli flora was demonstrated in 16 calves reared as dairy cow replacements. The incidence of antibiotic resistance among isolates, as measured by an Antibiotic Resistance Index (ARI), changed markedly with the age of the calf. The value was low initially, when the calves were 1–2 days old and housed with adult animals. It then rose rapidly during the first week after the animals had been weaned and moved into nursery pens. This change in ARI was associated with the isolation of strains resistant to four or five of the six drugs included in the sensitivity test. The ARI then fell from the third week to low levels by the time that the calves were five months of age. This fall was due to the isolation of an increasing proportion of sensitive E. coli strains. These differed from the sensitive strains which had colonized the calves in the early days of life so demonstrating that the change was not due to the reemergence of strains identified several weeks previously. The source of E. coli strains was presumed to be the calfs' environment but further investigations are required to prove this conclusively.     

The control of sulphate reduction in Escherichia coli by O-acetyl-l-serine

1. Extracts of Escherichia coli A.T.C.C. 9723 and K(12)703 contain serine transacetylase and O-acetylserine sulphhydrase. Synthesis of the latter enzyme is repressed by growth on l-cyst(e)ine and other sulphur compounds. 2. O-Acetyl-l-serine added to cells growing on glutathione or sulphate as source of sulphur induces the enzymes that catalyse (a) the activation of sulphate to adenosine 3'-phosphate 5'-sulphatophosphate (EC 2.7.7.4 and 2.7.1.25), (b) the reduction of adenosine 3'-phosphate 5'-sulphatophosphate to sulphite and (c) the reduction of sulphite to sulphide (EC 1.8.1.2). Hydrogen sulphide is liberated from cultures growing on sulphate as source of sulphur and in the presence of O-acetylserine. 3. The cysE mutants of E. coli K(12) lack serine transacetylase. Addition of O-acetylserine permits growth on sulphate as source of sulphur; at the same time the enzymes of sulphate reduction, previously absent, are synthesized. Such mutants have no detectable intracellular cyst(e)ine when starved of sulphur. 4. These results suggest that O-acetylserine is necessary for synthesizing the enzymes of sulphate reduction in E. coli. Its action does not appear to be by interference with the repressive control exerted over these enzymes by cyst(e)ine.     

Application of bacteriophages to control intestinal Escherichia coli O157:H7 levels in ruminants

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios > or = 10(2) terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10(10) PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be > or = 10(2). In addition, phages were maintained at 10(6) PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.     

Replication of bacteriophages in Escherichia coli mutants thermosensitive in DNA synthesis

Summary In E. coli mutants thermosensitive in DNA synthesis the capacity for replication of bacteriophages , P1 and T4 was studied in order to obtain more information about the biochemical lesions in such strains. Two mutant types were used. In one of them DNA synthesis stops immediately at the restrictive temperature (mutant 165/70). In the other type DNA synthesis continues at the elevated temperature for a residual time period before it comes to a halt (mutant 252). The thermolabile synthetic steps involved in both mutant types are presently still unknown.The temperate phages and P1 differ in their ability to replicate in the mutant types at temperatures non-permissive for host cell DNA synthesis. Replication of phage is blocked in 165/70 but can still take place in 252 after host DNA synthesis has come to a halt. Phage P1 shows the opposite behaviour. It grows in the mutant 165/70 but its ability to replicate in 252 at 42° C is restricted to the period of residual host cell DNA synthesis observed in uninfected cells. Replication of phage T4 on the other hand is unimpeded in both mutants at restrictive temperatures.     

The ecology of Escherichia coli in market calves fed a milk-substitute diet

Dynamic changes in the Escherichia coli population in the calf gut were studied over 21 days in a group of 18 intensively-reared market calves. Isolates were identified by O-serogrouping, biotyping and resistogram patterns. Seventy O-serogroups were identified among nearly 3000 E. coli isolates examined and these were subdivided into 416 strains by means of their biotype and resistogram. Seventy-five per cent of these strains were detected only once or twice, which points to the continual replacement of the E. coli flora with strains that showed low persistence in the gut. The rise in the frequency of antibiotic resistance observed during the study was not due to a change in the proportion of resistant to sensitive strains in the gut flora. It was a consequence of the displacement of the original flora by multiply-resistant strains, which presumably originated from the calves' environment.     

The ecology of Escherichia coli in market calves fed a milk-substitute diet

Dynamic changes in the Escherichia coli population in the calf gut were studied over 21 days in a group of 18 intensively-reared market calves. Isolates were identified by O-serogrouping, biotyping and resistogram patterns. Seventy O-serogroups were identified among nearly 3000 E. coli isolates examined and these were subdivided into 416 strains by means of their biotype and resistogram. Seventy-five per cent of these strains were detected only once or twice, which points to the continual replacement of the E. coli flora with strains that showed low persistence in the gut. The rise in the frequency of antibiotic resistance observed during the study was not due to a change in the proportion of resistant to sensitive strains in the gut flora. It was a consequence of the displacement of the original flora by multiply-resistant strains, which presumably originated from the calves' environment.     

Isolation and characterization of lytic bacteriophages against enterohaemorrhagic Escherichia coli

Aims: The objective of this study was to isolate, identify and characterize a collection of lytic bacteriophages capable of infecting enterohaemorrhagic Escherichia coli (EHEC) serotypes. Methods and Results: Phages were isolated from dairy and cattle feedlot manure using E. coli O157, O26 and O111 strains as hosts. Phages were enriched from faecal slurries by culture in 10× trypticase soy broth at 37°C overnight. Phage plaques were obtained by mixing the filtered culture supernatant with molten tryptone agar containing the phage E. coli host strain, pouring the inoculated agar on top of cooled TS agar and incubating the culture overnight. Phages were purified from plaques and screened against additional E. coli and EHEC strains by the efficiency of plating method (EOP). Phage CEV2, and five other phages previously isolated, were able to lyse all of the 15 O157 strains tested with EOP values consistently above 0·001. Two phages were found to be highly effective against strains of E. coli O157 through EOP tests and against O26 strains through spot tests, but not against the O serogroup 111 strains. A cocktail of eight phage that lyse E. coli O157 strains resulted in >5 log CFU ml^?1 reductions at 37°C. Multiplex‐PCR revealed that none of these eight phages carried stx1, stx2, hlyA or eaeA genes. Conclusions: A cocktail of bacteriophages was capable of lysing most strains of two EHEC serotypes. Significance and Impact of the Study: This collection of phages can be combined and potentially used as an antimicrobial cocktail to inactivate E. coli strains from O serogroups 157 and 26 and reduce their incidence in the food chain.