Aims: To examine Escherichia coli strains EQ1, DH5α, BLR and BL21 for known pathogenic mechanisms.
Methods and Results: Using specific DNA probes, the strains were shown not to carry the genes ...encoding invasion, various adhesion phenotypes or expression of a range of enterotoxins. The strains were unable to express long‐chain lipopolysaccharide and were susceptible to the effects of serum complement. Using a BALB/c mouse model, the strains were shown to be unable to survive in selected tissues or to persist in the mouse gut. Using a chick model, strains EQ1, BLR and BL21 invaded livers but not spleens; only strain EQ1 persisted in the chick gut. In Merino sheep, only strain EQ1 was detected 6 d post‐infection.
Conclusions: Escherichia coli strains EQ1, DH5α, BLR and BL21 did not carry the well‐recognized pathogenic mechanisms required by strains of E. coli causing the majority of enteric infections.
Significance and Impact of the Study: Escherichia coli strains EQ1, DH5α, BLR and BL21 were considered to be non‐pathogenic and unlikely to survive in host tissues and cause disease.
Newly hatched specific pathogen-free chicks were dosed with a suspension of
Bacillus subtilis spores prior to challenge with
Escherichia coli O78:K80, a known virulent strain associated with avian ...colibacillosis, 24
h later. A single oral inoculum of 2.5×10
8 spores was sufficient to suppress all aspects of
E. coli O78:K80 infection. Colonisation of deep organs was reduced by a factor of over
2
log
10
whilst colonisation of the intestine, as measured by direct caecal count, was reduced over
3
log
10
. Shedding of
E. coli O78:K80 was measured by semi-quantitative cloacal swabbing and was reduced significantly for the duration of the experiment, 35 days.
B. subtilis persisted in the intestine although with decreasing numbers over the same period. Challenge with the same dose 5 days after pre-dosing with spores overcame any suppressive effect of the spores.
Escherichia coli isolates were recovered from faecal samples taken from cattle, sheep and pigs at slaughter in England and Wales. Isolates (
n = 1227) selected at random from this collection were ...each hybridised in colony dot-blot experiments with an
eae gene probe that presumptively identified attaching–effacing
E. coli (AEEC). Of the 99 (8.1%)
eae positive isolates 72 were of ovine origin, 24 were of bovine origin and three of porcine origin. None were typed as O157:H7 whereas 78 were assigned to 23 serogroups and 21 were untypable. The most frequently isolated
eae positive serogroups were O156 (10), O26 (8), O103 (8), O108 (7) O56 (6) and O168 (6) of which serogroups O103 and O156 only were recovered from all three animal species. In tissue culture adherence assays, 36 representatives of
eae positive isolates of all serogroups and host of origin tested induced intimate attachment with varying degrees of actin accumulation and pedestal formation in the HEp-2 cells. The identity of the
eae type for these 36 was determined by specific PCR and the most prevalent intimin types were
eaeβ (15),
eaeγ (12) and
eaeε (4). Isolates were examined by PCR for the presence of other virulence determinants and five possessed
stx1 but none possessed
stx2. One O115
eaeε isolate possessed
cnf1 and 2,
hlyA,
etpD and
katP genes which is a novel combination of virulence determinants.
Escherichia fergusonii has been associated with a wide variety of intestinal and extra-intestinal infections in both humans and animals but, despite strong circumstantial evidence, the degree to ...which the organism is responsible for the pathologies identified remains uncertain. Thirty isolates of
E. fergusonii collected between 2003 and 2004 were screened using an
Escherichia coli virulence gene array to test for the presence of homologous virulence genes in
E. fergusonii. The
iss (increased serum survival) gene was present in 13/30 (43%) of the test strains and the
prfB (P-related fimbriae regulatory) and
ireA (siderophore receptor IreA) genes were also detected jointly in 3/30 (10%) strains. No known virulence genes were detected in 14/30 (47%) of strains. Following confirmatory PCR and sequence analysis, the
E. fergusonii
prfB, iss and
ireA genes shared a high degree of sequence similarity to their counterparts in
E. coli, and a particular resemblance was noted with the
E. coli strain APEC O1 pathogenicity island. In tissue culture adherence assays, nine
E. fergusonii isolates associated with HEp-2 cells with a ‘localised adherence’ or ‘diffuse adherence’ phenotype, and they proved to be moderately invasive. The
E. fergusonii isolates in this study possess both some phenotypic and genotypic features linked to known pathotypes of
E. coli, and support existing evidence that strains of
E. fergusonii may act as an opportunistic pathogens, although their specific virulence factors may need to be explored.
Escherichia coli O157 : H7 and Cryptosporidium parvum infections of man have been associated with direct contact with small ruminants. Colostrum protects neonates against gastrointestinal pathogens, ...and orphan lambs, which are common on petting farms, may be deprived of this protection. In a recent study, it was demonstrated that high shedding of E. coli O157 : H7 by an 8-week-old goat kid was associated with coincidental C. parvum infection. Furthermore, both pathogens were co-located in the distal gastrointestinal tract. It was hypothesized that colostrum deprivation and pre-infection with C. parvum predisposed young ruminants to colonization and increased shedding of E. coli O157 : H7. To test this, 21 lambs 5 weeks of age were divided into four groups as follows: (A) colostrum-deprived and inoculated with E. coli O157 : H7, (B) colostrum-deprived and inoculated with C. parvum and then E. coli O157 : H7, (C) conventionally reared and inoculated with E. coli O157 : H7, (D) conventionally reared and inoculated with C. parvum and then E. coli O157 : H7. C. parvum was detected between 8 and 12 days post-inoculation in most of the infected lambs. At 24 h post-inoculation with E. coli O157 : H7, all lambs were shedding between 5 x 10(4) and 5 x 10(7) c.f.u. E. coli O157 : H7 per gram of faeces. E. coli O157 : H7 was shed in higher numbers in the groups pre-inoculated with C. parvum, whether conventionally reared or colostrum-deprived. Interestingly, for the colostrum-deprived lambs on day 3, a significant difference in shedding of E. coli O157 : H7 was observed (P = 0.038), with the lambs inoculated with E. coli alone yielding higher counts than those pre-inoculated with C. parvum. From day 15 onwards, shedding of E. coli O157 : H7 was highest from the colostrum-deprived C. parvum-infected lambs, then (in descending order of shedding) the colostrum-deprived lambs, the conventionally reared lambs infected with C. parvum, and the conventionally reared animals. In total, four animals were euthanized, two at 24 h and two at 96 h post inoculation with E. coli O157 : H7 (two conventionally reared and two colostrum-deprived). All animals euthanized were from groups pre-inoculated with C. parvum prior to challenge with E. coli O157 : H7. On examination of tissues, in three of the four animals examined, multifocal attaching and effacing lesions were observed in the caecum, colon, rectum and at the recto-anal junction, and were confirmed by immunohistochemistry to be associated with E. coli O157 : H7.
To understand the role of flagella and fimbriae of Escherichia coli O78ratio K80 in avian
colibacillosis, day-old chicks were dosed orally with defined afimbriate and or aflagellate
mutants and ...colonization, invasion and persistence compared with that of the wild-type. In an
invasion model, chicks were dosed with 1 × 105 c.f.u. of a single strain and mutants defective
for type 1 fimbriae, curli fimbriae or flagella colonized livers by 24 h although the numbers of
bacteria present were significantly less than the wild-type. Mutants colonized between 50 and
75% of spleens whereas the wild-type colonized 100% of spleens. Additionally, the numbers of
mutant bacteria in colonized spleens were significantly less than the wild-type. Surprisingly,
mutants defective for the elaboration of more than one appendage were no more attenuated
than single mutants. In a persistence model, chicks were dosed with 1 × 102 c.f.u. of a single
strain and mutants defective for type 1 or curli or flagella or any combination thereof persisted
as assessed by cloacal swabbing for 5 weeks of the experiment less well than the wild-type. In
an additional persistence model, chicks were dosed with 5 × 102 c.f.u. of each of wild-type and
one mutant together. All mutants were significantly less persistent than the wild-type
(P < 0·001) and one mutant which lacked type 1, curli and flagella, was eliminated within 2
weeks. Analysis of the trends of elimination indicated that flagella contributed to persistence
more than curli, which contributed more than type 1 fimbriae. Here was evidence for a major
role in colonization, invasion and persistence played by type 1, curli and flagella.
The lipopolysaccharide of
Salmonella and other Gram negative pathogenic species has been implicated as a major virulence determinant and in this study we report the role of LPS of
S. Enteritidis in ...the colonisation and persistent gastrointestinal infection of young poultry. The gene encoding the unique O-antigen ligase,
waaL, was mutated by insertional inactivation in a well characterised
S. Enteritidis strain, S1400/94. The
waaL mutant, designated PCP, produced rough colonies on agar medium, did not agglutinate O9 antiserum, did not produce an LPS ladder on silver stained gels and was serum sensitive. PCP and a nalidixic acid marked derivative of S1400/94 (S1400/94 Nal
r) were used to orally challenge young chicks, separately and together in competitive index experiments. At post-mortem examination of 1-day-old chicks challenged S1400/94 Nal
r and PCP separately there were no significant differences in the numbers of S1400/94 Nal
r and PCP bacteria in tissues sampled on days 1, 2, and 5. By day 42 after challenge S1400/94 Nal
r bacteria were recovered in significantly higher numbers than PCP from the caecal contents (
P < 0.001). In competitive index studies in the 1-day-old chick PCP colonised, invaded and persisted in lower numbers than S1400/94 Nal
r. In 4-week-old chicks challenged separately, PCP bacteria were recovered from all tissues examined in significantly lower numbers than S1400/94 Nal
r. In competitive index experiments in 4-week-old chicks, PCP was not detected at any site and at any time point. Therefore, the O-antigen of
S. Enteritidis plays an important role in poultry infections although this role is less important in the newly hatched chick.
Escherichia coli O115 has been isolated from healthy sheep and was shown to be associated with attaching–effacing (AE) lesions in the large intestine. Following previous observations of interactions ...between E. coli O157 and O26, the aim of the present study was to assess what influence an O115 AE E. coli (AEEC) would have on E. coli O157 colonisation in vitro and in vivo. We report that E. coli O115- and O157-associated AE lesions were observed on HEp-2 cells and on the mucosa of ligated ovine spiral colon. In single strain inoculum, E. coli O115 associated intimately with HEp-2 cells and the spiral colon in greater numbers than E. coli O157:H7. However, in mixed inoculum studies, the number of E. coli O115 AE lesions was significantly reduced suggesting negative interference by E. coli O157. Use of the ligated colon model in the present work has allowed in vitro observations to be extended and confirmed whilst using a minimum of experimental animals. The findings support a hypothesis that some AEEC can inhibit adhesion of other AEEC in vivo. The mechanisms involved may prove to be of utility in the control of AE pathovars.
To investigate the role of fimbriae and flagella in the pathogenesis of avian colibacillosis, isogenic insertionally inactivated mutant strains of Escherichia coli O78:K80 strain EC34195 defective in ...the elaboration of type-1 and curli fimbriae and flagella were constructed by allelic exchange. Single and multiple non-fimbriate and non-flagellate mutant strains were compared to the wild-type in vitro in adherence assays with a HEp-2 cell line, a mucus-secreting cell line HT2916E, a non-mucus-secreting cell line HT2919A, tracheal explant and proximal gut explant. Mutant strains defective in the elaboration of type-1 fimbriae were significantly less adherent--in the order of 90% reduction--than the wild-type strain in all assays. Mutant strains defective in the elaboration of flagella were generally as adherent as the wild-type strain except when assayed with the mucus-secreting cell line HT2916E, for which a significant reduction of adherence--of the order of 90%--compared with the wild-type strain was observed. Mutant strains defective for the elaboration of curli fimbriae adhered as well as the wild-type strain in all assays, except when assayed in tests with gut explant tissue for which a significant reduction of adherence--of the order of 80%--compared with the wild-type strain was observed. Adherence to explants was to epithelial, not serous, surfaces and was 10-fold greater to tracheal than to gut explants. Together, these data support the hypothesis that type-1 fimbriae are significant factors in adherence, aided by flagella for penetration of mucus and curli fimbriae for adherence to the gut.
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