Showing papers on "Shigella dysenteriae published in 1989"

Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized P1 glycoprotein and production of cross-reactive monoclonal antibodies.

Abstract: Shiga toxin from Shigella dysenteriae 60R was purified to homogeneity by a novel one-step receptor analog affinity chromatography method. The method was based on the binding affinity of Shiga toxin for a specific disaccharide, Gal alpha 1----4Gal, which was also present in glycoproteins with P1 blood group seroreactivity produced in hydatid cysts from sheep infected with Echinococcus granulosus. Having shown that cyst fluid P1 glycoprotein bound Shiga toxin on a solid phase, a P1 glycoprotein affinity column was made by coupling P1-active substance to Sepharose 4B. Shiga toxin was purified by this method in large quantities (5 to 10 mg/20-liter batch) with a consistently good yield (greater than 80% of starting toxin). Shiga-like toxins I and II (SLT-I and -II, respectively) from Escherichia coli were also purified by the same method. A preparation containing SLT-II and SLT-I purified by receptor analog affinity chromatography was used to raise four monoclonal antibodies (MAbs) that were reactive with SLT-II by enzyme-linked immunosorbent assay. Three of these antibodies also reacted with Shiga toxin, which was the first clear demonstration of cross-reactivity between these toxins. One MAb, 4D1, which was specific for the B subunit of SLT-II and Shiga toxin, neutralized both toxins in a HeLa cell cytotoxicity assay. Two MAbs recognized the A subunit of both SLT-II and Shiga toxin by Western blot (immunoblot) analysis but were unable to neutralize either toxin. In addition, one B-subunit-specific MAb neutralized SLT-II alone, and a previously described Shiga toxin B-subunit-specific MAb was shown to be specific for Shiga toxin but not SLT-II.

Rapid method to detect shiga toxin and shiga-like toxin I based on binding to globotriosyl ceramide (Gb3), their natural receptor.

Abstract: Shiga toxin and the closely related Shiga-like toxins produced by Escherichia coli represent a group of very similar cytotoxins that may play an important role in diarrheal disease and hemolytic uremic syndrome. These toxins have the same biologic activities and according to recent studies also share the same binding receptor, globotriosyl ceramide (Gb3). They are currently detected, on the basis of their ability to damage several cell lines, by using expensive and tedious assays that require facilities for and experience with tissue cultures and are therefore most suitable for research laboratories. We have developed a rapid method to detect Shiga toxin and Shiga-like toxin I based on specific binding to their Gb3 natural receptor, which was coated onto microdilution plates. Bound toxin was then detected by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The sensitivity of the Gb3 ELISA was 0.2 ng (2 ng/ml) of purified toxin. The assay was positive with sonic extracts of Shigella dysenteriae serotype 1 strain 6OR (a Shiga toxin producer), E. coli serotype O26:H11 strain H30, and E. coli serotype O157:H7 (both Shiga-like toxin I producers). The assay was very specific in that no cross-reactivity was noted with purified cholera toxin, E. coli heat-labile and heat-stable enterotoxins, and Clostridium difficile cytotoxin, or sonic extracts of other cytotoxin-producing organisms, such as other shigellae, pathogenic and nonpathogenic E. coli, Salmonella spp., Campylobacter spp., and Aeromonas spp. These results were in complete agreement with a [3H]thymidine-labeled HeLa cell cytotoxicity assay and with detection of the structural genes by DNA hybridization studies with a Shiga-like toxin I probe. Quantitative analysis showed a high correlation between Gb3 ELISA and HeLa cell assay when fractions obtained at various stages of toxin purification were examined by both methods (r = 0.99, P < 0.01). This rapid Gb3 ELISA is sensitive and specific and may be diagnostically useful in cytotoxin-related infections.

Introduction and spread of multi-resistant Shigella dysenteriae I in Thailand.

Abstract: Outbreaks of Shigella dysenteriae I occurred in northeastern Thailand in the fall of 1986 and again in the spring and fall of 1987 for the first time in over 20 years. The epidemic strain of S. dysenteriae I was resistant to tetracycline, streptomycin, chloramphenicol, and trimethoprim-sulfamethoxazole, but susceptible to ampicillin. Trimethoprim resistance was chromosomally encoded by type I dihydrofolate reductase. In Ubon Province, where 10,000 cases of dysentery were reported, there were 3-5 cases of dysentery per 1,000 residents during the peak months, with 2-5 hospitalizations per 100 cases of reported dysentery. There were 2 deaths among 101 hospitalized, culture-confirmed cases. The overall case-fatality rate among reported cases of dysentery in this province was 0.9%. In contrast to S. flexneri infections, which occurred predominantly among children less than 5 years old, S. dysenteriae I infections occurred in all age groups. The large number of susceptibles appeared to be important in allowing rapid spread of S. dysenteriae I. In 1 village, 46% of 434 villagers reported dysentery; S. dysenteriae I was isolated from 24 out of 81 (30%) individuals cultured. Based on the prevalence of IgG antibody to S. dysenteriae I lipopolysaccharide, it was estimated that 76% of the villagers had been infected.

Cockroaches (Periplaneta americana) as carriers of agents of bacterial diarrhoea in Accra, Ghana.

Abstract: Bodies and intestinal contents of 208 cockroaches (Periplaneta americana), collected from kitchens in Accra and some surrounding villages were cultured for enteric bacterial pathogens. Six of them harboured three different serogroups of Salmonella, one had Shigella dysenteriae, 64 had Coliforms, 13 had Proteus species, two had Pseudomonas species and the rest (122) carried none of the bacterial species mentioned above. The presence of Salmonella species Shigella dysenteriae and Coliforms in these insects, which were collected from kitchens where foods are kept, points to the facts that these insects could play an important role in the transmission of these pathogenic organisms, especially in our environment. Permanent solution to these bacterial diarrhoea disease problems could only be solved when food, animals and the environments are free of these microbes.

Phosphate regulon in members of the family Enterobacteriaceae: comparison of the phoB-phoR operons of Escherichia coli, Shigella dysenteriae, and Klebsiella pneumoniae.

Abstract: The structure and function of the phoB and phoR genes of Shigella dysenteriae strains and Klebsiella pneumoniae, which are involved in regulation of the phosphate regulon, were analyzed. Complementation tests among the genes of Escherichia coli, S. dysenteriae strains, and K. pneumoniae for production of alkaline phosphatase indicate that S. dysenteriae serotype 2 and serotype 3 strains and K. pneumoniae are phoA+ phoB+ phoR+ but S. dysenteriae Sh and serotype 1 strains are phoA phoB+ phoR. Nucleotide sequences of phoB and phoR of S. dysenteriae Sh and K. pneumoniae are highly homologous to those of E. coli, except for a single base insertion found in phoR of S. dysenteriae Sh.

Antibiotics in the Management of Shigellosis in Children: What Role for the Quinolones?

Abstract: In the last 20 years, epidemic bacillary dysentery due to Shigella dysenteriae type 1 has become an important cause of serious morbidity and death in children less than 5 years of age in a number of developing countries; in many instances the infecting strains have been resistant to many or all of the usually recommended antibiotics. Moreover, an increasing proportion of Shigella strains of other serotypes isolated in all parts of the world are now resistant to the most commonly used antibiotics. The new fluoroquinolones have been shown to be active against Shigella in vitro and effective for the treatment of shigellosis in adults, but concern about the safety of quinolones has so far prevented the evaluation of these agents for the treatment of shigellosis in young children. Two observations, however, have led a Scientific Working Group of the World Health Organization to suggest that such studies would be appropriate for the following reasons: a one- or two-dose treatment is likely to be effective, and if so the amount of drug given would be far below the doses that have been determined to be toxic in animals; and nalidixic acid, which shares all the toxicity of the fluoroquinolones, is widely recommended--and in some areas routinely used--for the treatment of shigellosis in young children without reports of significant adverse effects. In the Group's view there is an urgent need to perform carefully conducted research to determine the safety and efficacy of fluoroquinolones for the treatment of shigellosis in children.

Causes of death and the histopathologic findings in fatal shigellosis.

Abstract: Thirty-seven children (median age, 2 years) with shigellosis in Bangladesh were subjected to postmortem examination to determine causes of death and the spectrum of intestinal histopathology. Infecting species were: Shigella dysenteriae 1, 7 cases; S. dysenteriae 2, 2 cases; Shigella flexneri, 23 cases; Shigella boydii, 4 cases; and mixed infection with Shigella boydii and Shigella sonnei, 1 case. Complicating conditions detected before death included malnutrition in 25 cases, pneumonia in 11 cases and septicemia in 8 cases. In all 37 cases the colon showed gross colitis, consisting of mucosal erythema and edema; superficial ulcerations were visible in 15 cases. Microscopically in the colon the lamina propria showed inflammatory cellular infiltration in 27 cases and crypt abscesses were present in 22 cases. In 9 cases each there were colonic glands in the submucosa and branching of colonic crypts, indicating increased regenerative activity of crypt cells. Severe lesions were mucosal denudation and deep ulceration in 15 cases with a pseudomembrane in 7 and pseudopolyposis in 2 of these patients. The most common underlying cause of death was colitis, whereas the most common immediate and associated causes were, respectively, septicemia and pneumonia. These results indicated that fatal childhood shigellosis results from severe colitis, often complicated by septicemia and concomitant malnutrition and pneumonia.

Congo red-mediated regulation of levels of Shigella flexneri 2a membrane proteins.

Abstract: The ability of Shigella spp. to bind Congo red from agar medium is generally correlated with their virulence properties. We used a metabolically active culture of Shigella flexneri 2a to determine the effect of Congo red on its membrane protein profiles. Virulent S. flexneri grown in the presence of Congo red at 37 degrees C showed increased levels of three proteins with Mrs of 43,000, 58,000, and 63,000 (43K, 58K, and 63K proteins) in the Sarkosyl-soluble membrane fractions. The observed phenomenon was temperature dependent. At 30 or 42 degrees C the protein levels remained unaffected by the presence of Congo red. Similar regulation of the levels of the 43K, 58K, and 63K membrane proteins was also observed with Shigella dysenteriae 1 and enteroinvasive Escherichia coli, but not with enteropathogenic E. coli. The cellular uptake of Congo red seemed to be essential, but not sufficient, for regulation. All three proteins reacted with human convalescent-phase sera in immunoblots of S. flexneri 2a Sarkosyl-soluble membrane fractions. Using the 43K-specific antiserum as the primary antibody, by indirect immunofluorescence studies, we detected an increase in the level of the 43K protein in S. flexneri which had invaded epithelial cells. These observations strongly indicate that the 43K, 58K, and 63K proteins are virulence associated. We propose that the observed regulatory effect of Congo red on membrane proteins of S. flexneri is mediated through induction. Since the same regulatory effect was also observed during the invasion of epithelial cells by S. flexneri, it is suggested that Congo red mimics some host tissue factor in vitro.

Clinical & bacteriological profiles of shigellosis in Calcutta before & after an epidemic (1984-87).

Abstract: Patients below 5 yr of age, hospitalised for shigellosis over a period of four years (1984-87), were studied. During the epidemic of bacillary dysentery (1984) isolation of different Shigella spp. as well as Shigella dysenteriae type 1 was high. Decreased isolation of Sh. dysenteriae type 1 and increased isolation of Sh. flexneri was observed during post-epidemic years (1985-87). Isolation of different Shigella spp. was always above 25 per cent from patients with dysentery and greater than 7 per cent from those with watery diarrhoea during the post-epidemic years. Higher incidence of shigellosis was observed amongst older children (greater than 3 yr). Most of the shigellosis patients complained of blood and mucus in stools. Vomiting was common among shigellosis patients presenting with watery diarrhoea whereas fever was commonly seen in patients with both dysentery and watery diarrhoea. Most patients of shigellosis presenting with blood and mucus in stools had no dehydration.

Hemagglutinating properties of Shigella dysenteriae type 1 and other Shigella species.

Abstract: Strains of Shigella dysenteriae type 1 cultured in Casamino Acids-yeast extract broth medium in the presence of 1 mM calcium chloride at 37 degrees C for 22 h induced hemagglutination of erythrocytes that was inhibited by N-acetylneuraminic acid, N-acetylneuramin-lactose, and alpha 1-glycoprotein. The hemagglutination was heat labile, and the absence of cell-surface appendages suggested a nonfimbrial adhesin(s). Under the same conditions, strains of Shigella flexneri (types 1a, 1b, 2a, and 2b) showed N-acetylneuraminic acid-resistant hemagglutination of erythrocytes.

Four new provisional serovars of Shigella.

Abstract: Four bacterial strains are described that possess the biochemical characteristics of Shigella species but do not belong to any of the established Shigella serovars or to any previously described provisional serovar. One strain fermented mannitol, and it is proposed that this be the type strain for a new provisional serovar of Shigella boydii. The remaining strains did not ferment mannitol and belonged to three different serovars. These strains are proposed as type strains for three new provisional serovars of Shigella dysenteriae. All four strains were invasive in a HEp-2 cell tissue culture test, but only one was invasive in the guinea pig eye test and might therefore be expected to cause dysenterylike illness in humans. It is important that the designation of such strains remain provisional until other reference laboratories have had the opportunity to search for additional isolates and the possible pathogenicity of these strains for humans can be further assessed.

Production of vero cytotoxin by Escherichia coli and Shiga toxin by Shigella dysenteriae 1 as related to the growth medium and availability of iron.

Abstract: Six strains of Escherichia coli producing Vero cytotoxin (VTEC) and six strains of Shigella dysenteriae 1 were examined for the production of extra- and intracellular Verocytotoxin (VT) and Shiga toxin respectively, in relation to the growth medium and availabilityof iron. VTEC secreted less extracellular VT1 or VT2 when grown in trypticase soy broth(TSB) containing the iron chelator desferal, as compared to bacteria cultured in iron repleteTSB. Growth in TSB containing desferal resulted in increased production of intracellulartoxin by VT1 producing strains of E. coli ; but had little effect on production of intracellulartoxin by VT2 producing strains. Both extra- and intracellular levels of Shiga toxin wereincreased by growth in TSB containing desferal. Combining intra- and extracellular toxin titres, strains of E. coli producing VT1 gavehighest titres following growth in TSB where iron was bound to the chelating agent desferal,while strains of S. dysenteriae 1 produced highest levels of toxin when grown in syncase-glucosebroth made iron depleted using Chelex-100. Strains of S. dysenteriae 1 did not growin syncase-glucose broth containing desferal.

Monoclonal Antibodies against the Surface Antigens of Shigella flexneri Serotype 1b and Shigella dysenteriae Serotype 1

Abstract: Monoclonal antibodies against the surface antigens of Shigella flexneri 1b and S. dysenteriae 1 were prepared. The specificities of the antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), and quantitative agglutination using microtiter plate. Monoclonal antibodies against S. flexneri 1b, designated Sf2B2 and Sf2G4, belonged to IgG2a and IgG1 subclass, respectively. The former was specific for S. flexneri 1b, whereas the latter was reactive not only to S. flexneri 1b, but also weakly to 3a and 4b. Monoclonal antibody against S. dysenteriae 1, Sd5E1 (IgM), reacted with S. dysenteriae 1, 3, 6, 7, and S. boydii 2.

Studies on Shiga-like toxin produced by enterohemorrhagic Escherichia coli : purification and characterization of the toxin and development of methods for identifying the toxin

Abstract: A simple purification method using DEAE cellulose column chromatography and immunoaffinity column chromatography was developed for purifying Shiga-like toxin produced by Escherichia coli O157:H7. About 0.75 mg of purified toxin was obtained from 5 liters of culture (62% recovery). The purified toxin was demonstrated to be immunologically, biologically and structurally indistinguishable from Shiga toxin. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detection of Shiga-like toxin. In the ELISA assay, Shigella dysenteriae type 1, Escherichia coli O157:H7 and some strains of Escherichia coli isolated from traveller's diarrhea were positive. Shiga toxin-resistant Vero cells were isolated by treatment of the cells with nitrosoguanidine. Immunofluorescence studies showed that the mutant Vero cells had lost toxin binding capacity. Samples of S. dysenteriae type 1 and E. coli O157:H7 showed cytotoxicity to the parent cells, but not to the mutant cells. Samples of other organisms showed either no cytotoxicity or cytotoxicity to both cell lines. The results suggested that (1) the presence of a receptor for Shiga-like toxin on Vero cells is essential for expression of cytotoxicity of the toxin, (2) the mutant Vero cells could be used to identify Shiga-like toxin producing organisms.

Relationship between multiple drug-resistance & enterotoxin production by Shigella species.

Abstract: Culture filtrates prepared with strains of Shigella dysenteriae 1 and those of Sh. flexneri and Sh. sonnei, collected from different geographical locations in India caused accumulation of fluid in rabbit gut loops, indicating their capability to produce Shiga and Shiga-like toxins respectively. Sh. boydii strains were noted for the first time to produce Shiga-like toxins. The failure of production of fluid accumulation in rabbit gut by Shigella strains that lacked the R-markers of the antibiotics tested and the gradual enhancement in the secretory response by strains containing R-markers in the increasing order indicate a possible correlation between acquisition of higher number of R-markers and enterotoxic activity. The non-transferable and non-curable nature of most of the drug resistance markers, coupled with the failure of the two transconjugants to elicit a secretory response and the capacity of the single cured variant to retain this property suggest the involvement of certain chromosomal locii in mediating drug resistance and enterotoxicity in Shigella.

Development of antibiotic resistance of type 1 Shigella dysenteriae strains (Shiga bacillus) isolated in Tananarive on the East coast of Madagascar

Abstract: From November 1988 to March 1989, 804 Malagasy children stools were studied and 37 Shigella strains isolated. 5 Shigella dysenteriae type 1 from Malagasy East coast (Mananjary), presented a multiply resistance to ampicillin, carbenicillin, streptomycin, chloramphenicol, tetracycline, sulphonamides and trimethoprim. This last resistance has recently appeared in this area.

Experimental studies on membrane permeability defects of drug-resistant bacteria.

Abstract: Multiply antibiotic-, lysozyme-and bacitracin-resistant, representative strains of staphylococci, Escherichia coli, Shigella dysenteriae and Klebsiella pneumoniae were compared respectively with their parent cultures in respect of their membrane permeability as judged by fluorescence polarization spectroscopy, using a sensitive fluorescent probe. The cytoplasmic membrane of the drug-resistant mutants was found to be less fluid and permeable, compared to that from the sensitive wild types. The study constitutes an attempt to determine the physical basis for the mechanism of resistance of certain bacteria against certain drugs.

The Shiga and Shiga-Like Cytotoxins: Gene Regulation and Functional Analysis of the Binding Subunits

Abstract: : Strains of Escherichia coli produce cytotoxins which are related to the Shiga toxin produced by Shigella dysenteriae type 1. Shiga-like toxin type I (SLT-I) and Shiga-like toxin type II (SLT-II) are produced by enterohemorrhagic E. coli which cause hemorrhagic colitis and/or the hemolytic uremic syndrome in humans . Shiga toxin, SLT-I, and SLT-II are primarily cell-associated cytotoxins that kill both Vero cells and HeLa cells in culture. A third SLT, the Shiga-like toxin type II variant (SLT-IIv), is produced by strains of E. coli responsible for the edema disease of swine. SLT-IIv, which is antigenically related to SLT-II, is markedly more cytotoxic for Vero than HeLa cells. structurally, all of these toxins are comprised of an A subunit, which is responsible for the enzymatic activity, and multiple B subunits which bind the toxin to a eucaryotic cell receptor.