Richard K.J. Luke

Inducible Plasmid-mediated Copper Resistance in Escherichia coli

The copper resistance in Escherichia coli determined by plasmid pRJ1004 is inducible. The level of resistance is proportional to the inducing dose of copper. The level of copper resistance in induced and uninduced cells changes with the growth phase of the culture. Induced resistant cells accumulate less copper than uninduced cells, so that reduced accumulation may be the mechanism of resistance. We propose that the inducible plasmid-coded copper resistance interacts with the normal metabolism of the cell to protect against toxic levels of copper while allowing continued operation of copper-dependent functions.

Oral administration of protease inhibits enterotoxigenic Escherichia coli receptor activity in piglet small intestine.

The virulence of enterotoxigenic Escherichia coli (ETEC) is attributed to their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa. A novel approach to preventing ETEC induced diarrhoea would be to prevent attachment of ETEC to intestine by proteolytically modifying the receptor attachment sites. This study aimed to examine the effect of bromelain, a proteolytic extract obtained from pineapple stems, on ETEC receptor activity in porcine small intestine. Bromelain was administered orally to piglets and K88+ ETEC attachment to small intestine was measured at 50 cm intervals using an enzyme immunoassay. K88+ ETEC attachment to intestinal sections that were not treated with bromelain varied appreciably between sampling sites. Variability in receptor activity along the intestinal surface is though to be caused by the localised effects of endogenous proteases. Oral administration of exogenous protease inhibited K88+ ETEC attachment to pig small intestine in a dose dependent manner (p < 0.05). Attachment of K88+ ETEC was negligible after treatment, resembling the levels of attachment of K88 to piglets of the genetically determined non-adhesive phenotype, which are resistant to K88+ ETEC infection. Serum biochemical analysis and histopathological examination of treated piglets showed no adverse effects of the bromelain treatment. It is concluded that administration of bromelain can inhibit ETEC receptor activity in vivo and may therefore be useful for prevention of K88+ ETEC induced diarrhoea.

The distribution and stability of Escherichia coli K88 receptor in the gastrointestinal tract of the pig

The levels of Escherichia coli K88 receptor were measured at various sites within the pig intestinal tract using an enzyme immunoassay. The amount of receptor in samples taken from K88-adhesive phenotype animals was found to vary widely along the length of the intestinal tract, but was usually highest in mucosal scrapings taken from the mid-small intestine. Receptor was evident in material collected near either end of the small intestine and was not apparent in material collected from the caecum or lower bowel. The ability of receptor-containing intestinal material to react with immobilized K88 adhesin was inhibited by exposure of the material to either trypsin or contents from the lower bowel, if the receptor-containing material was reacted with the immobilised K88 adhesin prior to exposure to trypsin or lower bowel contents, the bound material remained evident for 24 to 48 h. The possible implications of variable receptor activity in proteolytic environments in relation to pathogenesis and the determination of K88 phenotype in live pigs is discussed.

Bacteria, parasitic agents and rotaviruses associated with acute diarrhoea in hospital in-patient Indonesian children

Faeces from children (aged from one month to 12 years) with acute diarrhoea admitted to hospital in Yogyakarta, Indonesia, from June 1978 to June 1979, were examined for the presence of enteric pathogens. One or more recognized enteropathogens were identified in 56% of children. Rotaviruses were identified in 38% of all children. Toxigenic coliforms (predominantly Escherichia coli) were isolated from 12% of children. Salmonella sp. (6%), Shigella sp. (4%) and enteropathogenic parasites (predominantly Trichuris trichiura) from 3.5% of children. Mixed infections with two or more enteric pathogens were found in 7.6% of children. The incidence rate of each pathogen was correlated with age of the child, socio-economic level of the family and duration of breast feeding. Toxigenic coliforms were equally common in all age groups from both well-to-do and poor families. Enteropathogenic parasites appeared in increasing frequency with age. They were more common in artificially fed children and in children from families of low socio-economic level. The occurrence of multiple infection with mixtures of enteric pathogens increased with increasing age. Mixtures of parasites and other enteric pathogens only occurred in children with acute diarrhoea. These results provide baseline data about the relative importance of different enteropathogens in Indonesian children.

Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12. Four polypeptides involved in the conversion of 2,3-dihydroxybenzoate to enterochelin.

Four gene products involved in the enzymatic synthesis of enterochelin from 2,3-dihydroxybenzoate, L-serine and ATP (Luke, R.K.L. and Gibson, F. (1971) J. Bacteriol. 107,557-562; Woodrow, G.C., Young, I.G. and Gibson, F. (1975) J. Bacteriol. 124, 1-6) have been partially purified using a previously reported fractionation procedure (Bryce, G.F. and Brot, N. (1972) Biochemistry 11, 1708-1715). The products of genes E, F and G have been separated from each other and correspond to the E1, E2 and E3 activities described by Bryce and Brot. These three gene products were not completely separated from the product of gene D. We refer to these gene products as components E, F, G and D of the enzymic apparatus for biosynthesis of enterochelin. Certain properties and functions of the four semi-purified components have been investigated. The E component is involved in the activation of 2,3-dihydroxybenzoate and the F component in the activation of L-serine. The D component physically associates with the F and G components during gel filtration and chromatography on DEAE Sephadex. It is proposed that the synthesis of enterochelin from L-serine and 2,3-dihydroxybenzoic acid is catalysed in vivo by a multienzyme complex, enterochelin synthetase.

Serotypes of Escherichia coli in Sudden Infant Death Syndrome.

Aim:  To examine the diversity of Escherichia coli serotypes found in the intestinal contents of infants who died of Sudden Infant Death Syndrome (SIDS) compared with that in comparison infants.

Serotypes of Escherichia coli isolated from healthy infants in Berlin, Germany and Melbourne, Australia

The characterization of Escherichia coli strains isolated from healthy infants under one year of age with respect to O:H serotype, K1 and K5 antigens in two disparate parts of the developed world was the purpose of this investigation. A total of 450 strains were examined, 264 from Berlin and 186 from Melbourne. Of all the 220 different O:H serotypes found, 179 were only isolated once, 90 in Berlin and 89 in Melbourne. However, 30 of the 41 O:H serotypes (73.2%) found more than once were isolated in both centers. The most commonly identified serotypes were found in both centers and included O1:H-; O1:H7; O2:H2; O2:H4; O2:H7; O4:H5; O6:H-; O6:H1; O15:H1; O18:H7; O25:H1; and 075:H-. Potentially pathogenic serotypes were found in both cities. Enteropathogenic E. coli (EPEC)-associated serotypes (O18:H7; O26:H-; O44:H34; O86:H-; O128:H2) were present in 11 cases and enterohaemorrhagic E. coli (EHEC)-associated types including O26:H11; O128:H2) were present in four cases. The distributions of serotypes found were similar in the two cities, strongly suggesting the wider applicability of these results.

The isolation of cytotoxic necrotizing factor (CNF)-producing Escherichia coli from the intestinal contents of babies who died of Sudden Infant Death Syndrome (SIDS) and other causes as well as from the faeces of healthy babies

Strains of Escherichia coli producing cytotoxic necrotizing factor (CNF) have been isolated from intestinal contents of 16.8% of babies who died of Sudden Infant Death Syndrome (SIDS) and 16.5% of faeces from healthy babies. While no difference in CNF carriage was seen, it is noteworthy that these CNF-producing E. coli are present in such specimens. Some of the CNF-producing E. coli belonged to serotypes associated with this factor in other parts of the world.

Screening of pig intestines for K88 non-adhesive phenotype by enzyme immunoassay.

An adhesion test for binding of porcine brush border membranes to Escherichia coli cells that possess the K88 antigen (K88+) has been developed using enzyme immunoassay procedures. K88 pilus protein or K88+ E. coli cells were immobilized in the wells of polystyrene microtitre plates. These plates were incubated in the presence of material obtained by scraping the villous surface of pig small intestines. Adhesion of membrane material to immobilized K88 was detected by adding rabbit anti-brush border IgG followed by urease-labelled sheep anti-rabbit IgG conjugate. Action of bound enzyme on urea/bromo-cresol purple substrate solution (pH 4.8) produced an intense colour change from yellow to purple, enabling the test to be read visually. This test enables simple, rapid testing of large numbers of intestial samples and gives results that agree well with the more cumbersome microscopic adhesion test for adhesion of K88+ E. coli to purified brush border membranes.

Peptides specific for Mycobacterium avium subspecies paratuberculosis infection: diagnostic potential

Mycobacterium avium subspecies paratuberculosis (Map) is the causative agent of Johnes disease (JD). Current serological diagnostic tests for JD are limited by their sensitivity when used in sub-clinical stages of the disease. Our objective was to identify peptides that mimic diagnostically important Map epitopes that might be incorporated into a new-generation JD diagnostic. Four peptides were isolated from a phage-displayed random peptide library by screening on antibodies derived from Map-infected goats. The peptides were recognised by antibodies from Map-infected goats but not by antibodies from uninfected goats. The peptides elicited immune responses in rabbits, which reacted strongly with bona fide Map antigens proving the peptides were true epitope mimics. To assess the diagnostic value a panel of goat sera was screened for reactivitys with peptides. The peptides were recognised by antibodies from a proportion of goats infected with Map compared with control animals with a diagnostic specificity of 100% and the sensitivity ranged from 50 to 75%. Combinations of any two peptides improved sensitivity 62.5-87.5% and 100% sensitivity was achieved with three of the four peptides in combination. These data suggest peptides representing diagnostically important Map epitopes could be incorporated into a sensitive diagnostic test.