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The pathogenesis of Shigella diarrhea. VI. Toxin and antitoxin in Shigella flexneri and Shigella sonnei infections in humans.

Two strains of Shigella flexneri and one of Shigella sonnei were studied for toxin production in vitro. All of the three strains produced a cell-free cytotoxin that showed marked similarity to that produced by Shigella dysenteriae 1. Each toxin eluted in two distinct peaks on chromatography with Sephadex G-150, was destroyed by heating at 90 C for 30 min, and was neutralized by S. dysenteriae 1 antitoxin. Patients with infections due to S. flexneri and S. sonnei developed antibody that neutralized S. dysenteriae 1 toxin in vitro. In three of seven positive sera studied by sucrose density-gradient ultracentrifugation, antibody activity was associated only with the IgM fraction. The time course of the antibody response resembled that found in infections due to S. dysenteriae 1, in which an IgM antitoxin antibody has also been described. Since three species of Shigella have now been shown to be toxigenic, it is possible that bacterial toxin may play a role, along with bacterial invasion, in the pathogenesis of infections due to S. flexneri and S. sonnei, as well as those due to S. dysenteriae 1.

Antibody Formation

Studies in volunteers to evaluate candidate Shigella vaccines: further experience with a bivalent Salmonella typhi-Shigella sonnei vaccine and protection conferred by previous Shigella sonnei disease.

A bivalent vaccine consisting of Salmonella typhi strain Ty21a containing the 120 MDa plasmid of Shigella sonnei and expressing both S. typhi and S. sonnei lipopolysaccharides (LPS) on its surface was previously shown to protect significantly against S. sonnei disease in experimental challenge studies. However, protective efficacy could not be reconfirmed in volunteers with five subsequent lots of vaccine. One vaccine lot which resembled the initial protective lots of vaccine in biochemical and serological tests, and by electron microscopy, was administered to 16 volunteers who ingested three doses of 10(9) organisms each. Antibody secreting cells (ASC) specific for S. sonnei LPS were detected in the blood of 100% of vaccines, but no protection of these vaccines was demonstrated during a S. sonnei challenge study. To assess the ability of the volunteer model to detect infection-derived immunity, six volunteers who had had clinical shigellosis due to S. sonnei two months earlier were rechallenged with wild-type S. sonnei, together with 12 controls. Prior infection provided 100% protection against febrile illness (p = 0.05) and diarrhea (p = 0.04), thereby validating the volunteer model for assessing Shigella vaccines.

Administration, Oral

Superiority of MacConkey's agar over salmonella-shigella agar for isolation of Shigella dysenteriae type 1.

The efficiency of MacConkey's agar in the isolation of various types of Shigella was compared with that of salmonella-shigella (SS) agar during an extensive 18-month outbreak of disease caused by Shigella. In all, 1,580 isolates of Shigella were obtained from 12,307 rectal swab and fecal samples of patients with diarrhea and their contacts by direct plating onto MacConkey's and SS agars. Shigella dysenteriae type 1 and Shigella flexneri constituted 55 percent and 33 percent of all isolates, respectively, with a smaller number of Shigella boydii and Shigella sonnei. MacConkey's agar was superior to SS agar in the detection of S. dysenteriae type 1; 83 percent of the isolates were detectable on MacConkey's agar, compared with 40 percent on SS agar. In contrast, 84 percent of S. flexneri isolates were detectable on SS agar, compared with 51 percent on MacConkey's agar alone. These findings confirm that, for the culture of fecal specimens thought to contain S. dysenteriae type 1, one of the media used should be noninhibitory. Shigella was isolated at a consistently and significantly higher rate from fecal samples than from rectal swabs.

Bacteriological Techniques

Concentration of milk secretory immunoglobulin A against Shigella virulence plasmid-associated antigens as a predictor of symptom status in Shigella-infected breast-fed infants.

We conducted a prospective, community-based study of healthy breast-fed Mexican infants to determine the protective effects of anti-Shigella secretory IgA antibodies in milk. Milk samples were collected monthly, and stool culture specimens were obtained weekly and at the time of episodes of diarrhea. Nineteen breast-fed infants were found to have Shigella flexneri, Shigella boydii, or Shigella sonnei in stool samples. Ages of the 10 infants with symptomatic infection and the nine with asymptomatic infection did not differ significantly. Milk samples collected up to 12 weeks before infection were evaluated by enzyme-linked immunosorbent assay for secretory IgA antibodies against lipopolysaccharides of S. flexneri, S. boydii serotype 2, S. sonnei, and virulence plasmid-associated antigens. The geometric mean titers of anti-Shigella antibodies to virulence plasmid-associated antigens in milk received before infection were eightfold higher in infants who remained well than in those in whom diarrhea developed. The significance of milk secretory IgA directed against lipopolysaccharide was less clear. We conclude that human milk protects infants against symptomatic shigella infection when it contains high concentrations of secretory IgA against virulence plasmid-associated antigens.

Antigens, Bacterial

Detection of Shigella dysenteriae type 1 and Shigella flexneri in feces by immunomagnetic isolation and polymerase chain reaction.

A combination of immunomagnetic separation (IMS) and a polymerase chain reaction (PCR) procedure was used for direct isolation and identification of Shigella dysenteriae type 1 and Shigella flexneri from feces. Immunomagnetic particles were coated with monoclonal antibody MASFB, which is specific for a common epitope of the O polysaccharides of S. dysenteriae type 1 and S. flexneri. Bacteria bound to the beads were boiled in water, and target DNA was amplified with a primer pair specific for a gene coded on the invasion-associated locus (ial) of the large virulence plasmid of all four Shigella spp. and enteroinvasive strains of Escherichia coli. A 320-bp DNA fragment was generated and detected by an alkaline phosphatase-conjugated probe. Nonviable cells were also captured and detected by this technique. The method is simple and fast (7 h) and has a detection limit of ca. 10 Shigella organisms per g in fecal samples. The combined IMS-PCR assay correctly identified all 57 samples carrying S. dysenteriae type 1 and 68 samples carrying S. flexneri from 238 fecal specimens and also permitted detection of 17 samples carrying Shigella spp. from 113 specimens from diarrheal patients in whom shigellae were not detected by conventional culture.

Antibodies, Monoclonal

Immunochemical characteristics of Shigella sonnei and serotype 6 Shigella flexneri lipopolysaccharides and enterobacterial common antigen.

Immunochemical studies on Shigella sonnei and serotype 6 Shigella flexneri 0 antigens (lipopolysaccharides) and enterobacterial common antigen (ECA) isolated from Shigella sonnei were carried out. Oligosaccharide structure of 0-specific chain of serotype 6 Shigella fiexneri lipopolysaccharide was defined and beta-L-rhamnosyl-1,3-N-acetyl-D-galactosamin as immunodeterminant of type VI specificity was recognized. The structures of core regions of lipopolysaccharides isolated from R mutants of both Shigella subgroups were established. On the base of the serological and structural results it has been suggested that these core regions are identical and very close to RI core structure of E. coli C. The effective method of isolation and purification of enterobacterial common antigen from Shigella sonnei was elaborated and its immunological properties as well as chemical character defined.

Antigens, Bacterial

[ 771 Shigella strains isolated in a 15-year period from Americans living in Ankara. Comparisons with Shigella strains isolated from Turkish natives].

The author had worked as a bacteriologist in Ankara American Air Force Hospital from 1958 to 1972. During this period of time he was able to isolate 771 shigella strains from the American patients of the hospital. The amounts and the percentages of the subgroups are listed below together with the two other Ankara City Pediatric hospital results (see article). american community lived in Ankara for 15 years and shared many aspects which were important in shigella epidemiology like using city's tap water, shopping from the same green groceries to by fruits and vegetables so on. For shigella cases Ankara had seasonal alterations in July and August being the peak months. Americans, disease followed that course exactly. The big difference was being the local people of the city had those epidemics with the flexneri strains but Americans had them with the sonnei strains. To show the possible sources which could infect the community time to time and make sonnei predominant yielded negative result. All the patients stools were cultured. Sensitivity studies employing "plate disk diffusion" techniques were done. All patients treated accordingly. Stools checked for routine carriership exams. None become a chronic carrier. American installations had their shops, clubs, mess halls. The food handlers of these places had checked routinely by the preventive medicine office. None found infected. Only at 1963 the amount of flexneri isolations were higher than sonnei. All the remaining years sonnei were predominant. Antibiotic sensitivity studies showed no differences between subtypes. Except streptomycin and sulphamide strains stayed fairly sensitive to other antibiotics. Resistance factors contained maximum 6 markers. The little Americans community in Ankara consisted a unique and highly socioeconomic-wise developed group of the city. Many attempts to find the answer for the question is "who is infecting who" was always fruitless. That gives the impression as if the Americans were more sensitive to the shigella infections caused by sonnei strains for some reason. In another words Americans were more resistant to the infections other than sonnei.

Dysentery, Bacillary

Effect of prior infection with virulent Shigella flexneri 2a on the resistance of monkeys to subsequent infection with Shigella sonnei.

All virulent shigellae have large plasmids. Plasmid-associated genes encode the expression of membrane-associated proteins (MAP), some of which correlate with the ability to invade susceptible epithelial cells. These MAP are serologically related in all of the shigella serotypes and evoke an antibody response after infection. To determine whether the MAP have a significant role in protection, 24 monkeys were infected with virulent Shigella flexneri 2a. After recovery, one group (with controls) was rechallenged with S. flexneri 2a; another group (with controls) was fed Shigella sonnei. The animals that were rechallenged with S. flexneri 2a were protected, while those that were fed S. sonnei experienced the same incidence of disease as controls. No differences in serum immune response to MAP after primary infection with S. flexneri were detected in immunoblots using lysates of S. flexneri or S. sonnei or in ELISA using water extracts of these strains.

Animals

Evaluation of two colored latex kits, the Wellcolex Colour Salmonella Test and the Wellcolex Colour Shigella Test, for serological grouping of Salmonella and Shigella species.

Two colored latex kits (the Wellcolex Colour Salmonella Test [WCT-Salmonella] and the Wellcolex Colour Shigella Test [WCT-Shigella]; Division Diagnostics, Laboratories Wellcome S.A., Paris, France), which allow identification of the most frequently encountered Salmonella serogroups and Shigella species, respectively, were evaluated. WCT-Salmonella and WCT-Shigella yielded sensitivities of 98.4 and 98%, respectively, and a specificity of 100% when they were tested on pure cultures received at a reference laboratory.

Evaluation Studies as Topic

Site-specific recombinase genes in three Shigella subgroups and nucleotide sequences of a pinB gene and an invertible B segment from Shigella boydii.

Inversional switching systems in procaryotes are composed of an invertible DNA segment and a site-specific recombinase gene adjacent to or contained in the segment. Four related but functionally distinct systems have previously been characterized in detail: the Salmonella typhimurium H segment-hin gene (H-hin), phage Mu G-gin, phage P1 C-cin, and Escherichia coli e14 P-pin. In this article we report the isolation and characterization of three new recombinase genes: pinB, pinD, and defective pinF from Shigella boydii, Shigella dysenteriae, and Shigella flexneri, respectively. The genes pinB and pinD were detected by the complementation of a hin mutation of Salmonella and were able to mediate inversion of the H, P, and C segments. pinB mediated H inversion as efficiently as the hin gene did and mediated C inversion with a frequency three orders of magnitude lower than that of the cin gene. pinD mediated inversion of H and P segments with frequencies ten times as high as those for the genes intrinsic to each segment and mediated C inversion with a frequency ten times lower than that for cin. Therefore, the pinB and pinD genes were inferred to be different from each other. The invertible B segment-pinB gene cloned from S. boydii is highly homologous to the G-gin in size, organization, and nucleotide sequence of open reading frames, but the 5' constant region outside the segment is quite different in size and predicted amino acid sequence. The B segment underwent inversion in the presence of hin, pin, or cin. The defective pinF gene is suggested to hae the same origin as P-pin on e14 by the restriction map of the fragment cloned from a Pin+ transductant that was obtained in transduction from S. flexneri to E. coli delta pin.

Amino Acid Sequence

A case of shigellosis caused by Shigella dysenteriae type 1 and Shigella flexneri type 5B.

Shigella dysenteriae type 1 and Shigella flexneri type 5b strains were isolated as causative agents of bacterial dysentery in a patient having visited South-East Asia. Both strains are a rare finding for Bulgaria. S. dysenteriae 1 strains have not been isolated since 1962, and there were only single isolates of S. flexneri 5b. The strains were of the same antibiotic resistance patterns. Conjugation experiments showed that resistance is determined by transferrable R-plasmids having identical characteristics. It is assumed that in the patient's gut transfer of an R-plasmid occurs from E. coli of the normal flora to the pathogenic shigellae.

Bulgaria

Studies with a new generation of oral attenuated shigella vaccine: Escherichia coli bearing surface antigens of Shigella flexneri.

In an attempt to develop a safe, proliferating, oral, attenuated vaccine against shigellosis, genes that control the synthesis of group- and type-specific somatic antigens of Shigella flexneri 2a were transferred via conjugation to a recipient strain of Escherichia coli. The resultant hybrid (E. coli expressing shigella surface antigens) vaccine strain, PGAI 42-1-15, believed to have a complete (smooth) lipopolysaccharide, was given to volunteers in two vaccination-challenge studies. The vaccine was well tolerated and gave evidence of intestinal proliferation. In trial no. 1, volunteers given two doses of vaccine one month apart were challenged after eight weeks with 10(4) virulent S. flexneri 2a. Attack rates were comparable in vaccinees (50%) and controls (40%). In trial no. 2, vaccinees were given three weekly doses of vaccine and were challenged four weeks later with a small inoculum (10(2)) of S. flexneri 2a. Again, attack rates among vaccinees (47%) and controls (39%) were similar. It is unclear why this theoretically ideal, live shigella vaccine failed to protect against S. flexneri 2a.

Administration, Oral

Comparison of Hektoen and Salmonella-Shigella agar on 6033 stools of human origin submitted for routine isolation of Salmonella sp. and Shigella sp.

From 1989 through 1991, 6033 stool specimens from patients at Orleans Hospital (France), were cultured for enteric pathogens. Hektoen agar (HE) was compared with Salmonella-Shigella agar (SS) after direct streaking and after overnight enrichment in Mueller-Kauffmann broth. Yersinia strains were not computed. Seven strains of Salmonella serotype Typhi were isolated, on both media, and excluded from computations for discussed reasons. Out of a total of 9 Shigella strains, HE isolated 9 and SS isolated 7. No statistically significant difference appeared about isolation of non-Typhi Salmonella strains (127 vs 114 from direct streaking, 237 vs 243 after enrichment). Predictive values of suspicious colonies were not statistically different. Numbers of false positives were not different, except for black centred colonies of the direct streaking (231 on HE vs 186 on SS, p less than 0.05). The skill of technologists was not taken into account but was found likely to be more accurate on SS agar. This data suggest that, since the frequency of Shigella sp became much lower, the differential of efficiency of HE vs SS agar is no longer obvious in human medical routine.

Agar

Live Shigella flexneri 2a and Shigella sonnei I vaccine candidate strains with two attenuating markers. I. Construction of vaccine candidate strains with retained invasiveness but reduced intracellular multiplication.

Live Shigella flexneri 2a and Shigella sonnei Phase I vaccine candidate strains with two virulence-reducing markers were constructed through stepwise incorporation of weakly attenuating purine auxotrophy with subsequent rifampicin-resistance (RNA polymerase) mutation to yield optimal attenuation. These vaccine candidate strains showed an unaltered plasmid profile; did not cause keratoconjunctivitis in the Sereny test, while being excreted for a short but still marked period and providing partial protection from wild-strain infection; exhibited for guinea-pig conjunctival epithelia, HeLa cells and rat enterocytes a maintained invasiveness with reduced intracellular multiplication with little, if any, reversible cell damage; and produced, just as their ultrasonic lysates, no exudative reaction in the rabbit gut loop test.

Animals

Live Shigella flexneri 2a and Shigella sonnei I vaccine candidate strains with two attenuating markers. II. Preliminary results of vaccination of adult volunteers and children aged 2-17 years.

Shigella flexneri 2a and Shigella sonnei I vaccine candidate strains were tested for tolerance in 14 adults as well as for tolerance and immunogenicity in children aged 2-17 years. For fresh culture material, the limit for side effects fading within 36 h such as meteorism, loose stools, tenesmus and slight temperature, and tolerated by volunteers was 3 x 10(9) colony-forming units (c.f.u.). Excretion of vaccine strain germs for 2-3 days was observed only at doses of 10(10) c.f.u. and greater (2-3 x 10(10)). Children tolerated up to the maximum dose of 1-3 x 10(9) c.f.u. of the lyophilized vaccine without side effects. No vaccine strain germs were found in faeces, antigen being detected for 4-5 days. The total dose of 1-3 x 10(9) c.f.u. being distributed among three oral inoculations at intervals of 1-2 days induced coproantibodies in 70% of the vaccinees during the first 3 months, and in 30-40% up to 6 months. In contrast, the single application of this total dose for primary immunization, or booster after 10 months, proved to be more efficient. Ninety per cent of the subjects had markedly elevated titres for not less than 6 months.

Adolescent

Construction and evaluation of live attenuated vaccine strains of Shigella flexneri and Shigella dysenteriae 1.

Shigellosis is an invasive disease of the human colon which is particularly prevalent among children of the developing world. No proper vaccine is available to protect against this enteric disease. It is currently accepted that only live strains with attenuated virulence administered orally may elicit protective immunity at the level of the colonic mucosa, which is the exclusive site of multiplication of causative microorganisms such as Shigella flexneri and Shigella dysenteriae 1. We have constructed such vaccine candidates based on the destruction of virulence genes responsible for selected steps of the infection process. In S. flexneri, a combination of two mutations impairing cell-to-cell spread (icsA) and aerobactin production and transport (iuc, iut) which support growth within tissues provide a well tolerated and protective vaccine prototype against shigellosis in macaque monkeys. In S. dysenteriae 1, similar mutations are currently being introduced, in addition to one which eliminates the catalytic activity of Shiga toxin. These mutants and others will be tested soon in human phase I trials.

Animals

Biological properties of Shigella flexneri 2A toxin and its serological relationship to Shigella dysenteriae 1 toxin.

A toxin extracted from heat-inactivated, alkaline-treated Shigella flexneri 2a showed biological properties similar to those of Shigella dysenteria 1 toxin. The S, flexneri 2a toxin was lethal to mice, enterotoxic for ileal loops of rabbits, and cytotoxic for HeLa cells. A serological relationship between S. flexneri 2a and S. dysenteriae 1 toxin was shown with cross neutralization tests.

Antigens, Bacterial