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Virulent form I Shigella sonnei strains contain a 120-megadalton plasmid that is absent in their form II derivatives, which are always avirulent and devoid of O side chains. In the present study, 165 biochemical and antibiotic traits were assessed, but no experimentally useful phenotype could be associated with this large form I plasmid. Therefore, the form I plasmids of several S. sonnei strains were tagged with the antibiotic resistance transposons Tn3, Tn5, or Tn10. Transposon-tagged form I plasmids were not self-transmissible, but could be mobilized by the plasmid R386. Form II S. sonnei transconjugants for the form I plasmid acquired both virulence and the ability to synthesize form I antigen, establishing that these properties are plasmid mediated. Further studies indicate that this 120-megadalton form I plasmid is physically unstable in any of several host bacteria and suggest that it is a member of the FI incompatibility group. Also, two commonly observed, small plasmids of S. sonnei, of 3.2 and 3.9 megadaltons, were shown to encode either colicin E1 production or resistance to streptomycin and sulfonamide, respectively.

The most essential differences in the time course proliferation of virulent and a virulent Sh. sonnei and Sh. flexneri strains in the pulmonary model were found to become, manifest 3--6--9 hours postinoculation. In contrast to the avirulent Sh. flexneri strains smooth type, with its intensive elimination, the avirulent Sh. sonnei strains in phase I were preserved on their primary infective level during these hours, and their subsequent elimination was slower. The differences thus revealed seem to confirm the opinion that Sh. sonnei (phase I) antigen is a type C (capsular) antigen having pronounced antiphagocytic activity.

IgG antibody response to antigens coded by the virulence plasmid of enteroinvasive pathogens was studied in dysenteric patients by ELISA. A plasmidless Escherichia coli K-12 strain and its transconjugant harbouring the 140 megadalton plasmid of a virulent E. coli 0124 strain were used as antigens. Sera of 12 dysenteric patients taken on the first, third an twelfth week after Shigella sonnei infection were examined. For comparison sera of 23 nondysenteric persons were also applied. Titres of the first week samples did not differ significantly from those of the controls. Patients' sera taken on the third week showed a fivefold rise in titres as compared to the first week ones. Antibody activity remained on a comparable high level till the twelfth week. The data prove that there is a considerable IgG response to plasmid coded antigens common in shigellae and enteroinvasive E. coli.

The outer membrane proteins of intact S. sonnei cells of a strain virulent in the keratoconjunctival test contain up to 24 polypeptides; one of them (with a molecular weight of about 70 KD) is absent in similar preparations of avirulent nonpenetrating shigellae in phase I, genetically related to the above strain. Study of the composition of the outer membrane proteins of S. sonnei cells in phase II has revealed that these cells, when compared with smooth bacteria, show qualitative and quantitative differences in their polypeptide fractions; of these, three fractions are characteristic of rough bacteria.

The data obtained in this investigation confirm that the modified indirect enzyme immunoassay (EIA) permits the differentiation of virulent bacteria of the genus Shigella and enteroinvasive Escherichia (group 1), regularly containing virulence plasmids with a molecular weight of 120-140 MD, from their avirulent variants which have lost these plasmids (group 2). The ratio of the optic density (OD) values of the positive control samples (the OD of group 1) to the OD values of the negative ones (the OD of group 2) is significantly higher than 2.1. As revealed by EIA, the differences between groups 1 and 3 (avirulent Shigella strains and E. coli smooth strain O124, retaining high-molecular plasmids with a molecular weight of 120-140 MD or their fragments) are statistically insignificant. The ratio of the OD of group 1 to the OD of group 3 is significantly less than 2.0. Analysis of outer membrane protein (OMP) preparations isolated from S. flexneri virulent strain 2a and its isogenic avirulent plasmid-containing variant has revealed significant differences in their EIA results. The ratio of the OP of OMP preparations isolated from the virulent strain to the OD of OMP preparations from the avirulent strain exceeds 2.1.

A novel transposon mutagenesis system has been developed for Shigella. We have used it to isolate specific mutants of Shigella sonnei and Shigella dysenteriae 1 that fail to produce lipopolysaccharide 'O' side chains. The virulence of the mutants was evaluated in the Sereny test and in a HeLa cell invasion assay. All HO'-minus mutants failed to provoke a positive Sereny reaction but retained the ability to invade HeLa cells. This demonstrates that 'O' side chains are virulence factors of S. sonnei and S. dysenteriae 1. 'O'-minus mutants of S. sonnei which still contained the Form I plasmid were capable of invading HeLa cells whilst plasmid-minus mutants were not, demonstrating that this plasmid encodes properties other than 'O'-antigen production that are involved in virulence.

The authors present the results of a comparative study of Sonne dysentery and of the intestinal coli infection 0124 and 0151; significant differences in their epidemiology shown were chiefly caused by a different degree of pathogenicity of the causative agents of these diseases. A low degree of escherichia pathogenicity, and, in this connection, dependence of the character of the infection manifestation on the dose of the causative agent indicated food factor of the infection spread to be necessary for the origination of the clinically expressed disease. Epidemiological observations and bacteriological data showed that in diseases of escherichia etiology only those persons who had clinical manifestations of the disease could serve as epidemiologically significant sources of infection. In this connection prophylaxis of the mentioned affection should be primarily based on the intensification of the general sanitary measures.

Comparison of polyacrylamide gel electrophoretic protein profiles of four isogenic sets of virulent phase I Sh. sonnei strains and their avirulent phase I cells revealed no differences in outer membrane protein composition between virulent and avirulent derivatives. However, significant qualitative and quantitative differences were found in composition of major outer membrane proteins between strains of different origin. All four strains tested contained one major protein of 33K. This protein was susceptible to proteolytic enzymes and was found to be heat modifiable. Other major proteins of 35K and 37K present in three strains and 36K present in one strain were identified as peptidoglycan associated proteins.

The results obtained in the study of the biological activity of the lysates of 14 Sh. flexneri and Sh. sonnei strains differing in their virulence are presented. In the lysates of the virulent cultures 2 kinds of biological activity corresponding to the fractions of peaks I and II could be detected: one fraction possessed HeLa toxicity, the other fraction possessed enterotoxic activity. The avirulent nonpenetrating mutants had no HeLa toxicity, but retained their capacity for producing factors similar to enterotoxin. When added to a nontoxic dose, HeLa toxin produced by the virulent Shigella strains enhanced the penetration capacity of the virulent strains and imparted this capacity to the avirulent cultures of a homologous Shigella species.

The authors conducted an immunochemical analysis of the surface soluble antigen preparations (SSAP) obtained by extraction with physiological saline of the virulent Sh. sonnei microbes killed with acetone. Immunochemical studies by the immunoelectrophoresis and immunodiffusion methods in agar gel, separation on sepharose 4B and in polyacrylamide gel showed the presence of 3 groups of the antigens in the SSAP composition: 1) protein antigens, determining the commonness with the Sonne microbes of the II phase and intergenera associations with typhoid and enteropathogenic E. coli; 2) specific somatic antigen of the Boiven type present in small amounts in the SSAP; 3) specific antigens differing by specificity from the Boiven antigen detected in the microbes of the I phase only and possibly playing a role in the virulent and immunogenic properties of the causative agent.

Virulent Sh. flexneri strain 2a, Sh. sonnei strain, attenuated Sh. flexneri vaccine strain 2a 516M, and Sh. sonnei vaccine strain 6S (isolated by Yu. A. Belaya), as well as streptomycin-dependent Sh. flexneri strain 2a 1605/3 (isolated by V. V. Sergeev) were introduced into the ligated loops of the rabbit ileum. The use of light and immunofluorescent microscopy, the measurement of the volume of the fluid in the intestinal loops and the quantitative inoculation of their contents resulted in revealing the differences in the properties of the virulent and vaccine strains. The vaccine strains, in contrast to the virulent strains, did not proliferate in the lumen and did not cause the accumulation of fluid in the intestinal loops. They retained sharply limited, especially in the streptomycin-dependent bacteria, ability to penetrate into enterocytes and, via their cytoplasm, into the basement membrane, but lost their ability to proliferate in the cytoplasm of enterocytes (and probably even deteriorated there) and to cause plurulent ulcerous inflammation. This indicates that vaccine strains have insignificant residual virulence and suggests that the intestinal loop models, together with other models, may be used for testing the safety of vaccines prepared from Shigella strains.

Virulent Shigella sonnei synthesize a surface antigen (form I) which appears to be one of several requirements needed for this host to invade epithelial cells. Upon restreaking on agar media, form I cells readily and irreversibly generate form II cells that lack the form I antigen. All form II cells are avirulent. Plasmid deoxyribonucleic acid of form I and II cells of four different S. sonnei isolates, obtained from different areas of the world, was analyzed by agarose gel electrophoresis. A large plasmid (approximately 120 megadaltons in three of the strains) that is present in form I cells was always absent from form II derivatives. Attempts to transfer conjugally only this large plasmid from form I to genetically marked form II cells were unsuccessful. However, a composite molecule, apparently formed by recombination between the large form I plasmid and a self-transmissible plasmid, was found to transfer the form I trait. Transconjugant S. sonnei strains acquiring the form I antigen could retransfer this trait to S. sonnei, Shigella flexneri, or Salmonella typhi. These preliminary findings demonstrate that S. sonnei form I antigen synthesis is mediated by a large plasmid which is lost spontaneously at a relatively high frequency from S. sonnei strains.

Shigella sonnei lipopolysaccharide (LPS), either in phase I (extracted from the smooth strains), or in phase II (extracted from the rough strains) reduced mean arterial pressure and heart rate following a 640 micrograms/Kg intravenous (iv) bolus injection in the urethane-anesthetized rat. Lower doses were without significant effects. Escherichia coli LPS (640 micrograms/Kg iv) produced a greater and more prolonged hypotension, however the negative chronotropic effect was less severe. The iv injection of the lipid A moiety of the LPS molecule of Shigella sonnei resulted in a more pronounced hypotensive effect than that obtained with the parent LPS, thus showing that lipid A is the vasodilatating fraction of the molecule. When lipid A was injected in rats pretreated with aspirin or with naloxone, the hypotensive effect was significantly reduced and the decrease in heart rate was reverted in tachycardia. In atropine-pretreated, surgically bivagotomized rats, iv injection of lipid A was followed by a long-lasting pressor effect. The date indicate that Shigella sonnei endotoxin has cardiovascular effects, mainly due to its lipid A moiety of the molecule. The hypotensive effect involves a wide range of mechanisms.

Two distinct regions required for both contact hemolysis and entry into LLC-MK2 cells were cloned into Escherichia coli from the Shigella sonnei form I plasmid, pSS120. The first region was cloned into an E. coli HB101 strain containing noninvasive Tn1 insertion mutants of the form I plasmid, and expression of ipa (invasion plasmid antigen) gene products was restored. The plasmid carrying the first region was then transformed into E. coli lacking the form I plasmid, and additional DNA fragments from the form I plasmid were cloned into the same recipient on compatible vectors. Five of these double transformants were found to be positive for contact hemolysis activity. Restriction analysis of these five clones indicated that the previously reported ipa locus and the invA locus were present on the second plasmid region. Only the strains carrying both of these regions were active in contact hemolysis and cell invasion assays. Several proteins, including the a, b, c, and d proteins encoded by the ipa genes, were detected in the double transformants by Western blot (immunoblot) analysis with serum of a monkey convalescing from shigellosis. A positive regulator was suggested to exist in the first region, since the amounts of most of these proteins were simultaneously increased in the presence of this region. Subcloning and nucleotide sequencing indicated that this positive regulator gene was virF. Product analysis of the virF gene with minicells showed that two peptides (30 and 21 kilodaltons) were synthesized and that at least the 30-kilodalton protein was essential for regulation of the ipa genes.

Possible ranges of virulence of Sh. sonnei strains isolated from the patients and carriers in the foci of Sonne dysentery, supposedly with the same source of infection, were studied. Sh. sonnei strains isolated from carriers proved to be less virulent than the strains isolated from patients with the clinical manifestation of the infection. A hypothesis was put forward explaining some aspects of carrier state in Sonne dysentery, which most probably was explained by the refracterity of the macroorganism, not by the alterations in the genom of the causative agent.

In experiments on human fetal intestinal explants infected with shigellae the specific multiplication rate of these infective agents, found to be 0.026, and the maximum level of their accumulation in erythrocytes, reaching 22-36 microbes per cell, have been determined. These phenomena can be observed after at least 3-hour incubation and end in the release of the infective agents from the affected area with shedding epithelial elements (villi). Shigellae, aggregated in the shed villi easily adhering to the unaffected mucosa, ensure the intensive invasion of the epithelium, which leads to the continuation of the process. The regularity thus revealed indicates that the population cycle of the development of shigellae is limited by short intervals of 3-4 hours. During these intervals the repeated invasion and the release of shigellae, together with the shed epithelium, into the chyme-containing intestinal cavity occur. The conditions for the multiplication of shigellae and their specific multiplication rate in chyme are minimal (0.016).

Experiments in mice have demonstrated that in the presence of influenza infection the suppressing effect of virulent shigellae on the secondary humoral and cell-mediated immune response is enhanced. Under such conditions avirulent shigellae can also show suppressing effect which is usually absent in these organisms.

A case of Shigella sonnei bacteremia in a 65-year-old patient suffering from diabetes mellitus is discussed. The isolated strain had plasmid-mediated serum resistance and excreted aerobactin. The presence of diabetes mellitus as an underlying disease and the production by the Shigella sonnei isolate of factors related to bacterial invasion may have contributed to the bacteremic episode.

All enteropathogens first attach to the glycocalyx on the enterocyte surface. Then noninvasive cholera vibrios and enterotoxicogenic E. coli multiply on the enterocyte microvilli producing enterotoxins which cause pronounced enterocyte hypersecretion. Invasive enteropathogens differ in the model of penetration into enterocytes, multiplication site, the effect on the fate of invaded enterocytes, the ability of the infectious process for generalization. Recent studies have confirmed the decisive role of Shigella intraepithelial multiplication in the development of ulcerative dysenteric mucosal inflammation, Salmonella penetration through the enterocytes, multiplication in macrophages and development of not only enterocolitis but generalized infection as well. In case of enteropathogenic E. coli causing infantile enteritis, multiplication of the attached germs with the resulting damage to the enterocyte surface plays the main role whereas bacterial invasion through the enterocytes is limited and of secondary importance. All the invasive enterobacteria produce peculiar cytotoxic enterotoxins which stimulate but also damage the enterocytes.

The authors present the results of study of the virulence of shigellae isolated from carriers and patients suffering from acute dysentery, on a continuous culture of E1 and Hep-2 cells. The virulence of shigellae isolated from carriers displayed no significant difference from the virulence of shigellae isolated from patients with a mild and moderately severe forms of dysentery. In the patient's organism shigellae were capable of retaining the initial virulence for a long time, despite the treatment and the influence of the macroorganism's protective factors. The authors believe that this was connected with the capacity of Shigellae to parasitic life in the cells of human intestinal epithelium, and, apparently, played a definite role in the formation of carrier state.

Proper view on the true prevalence of Sonne dysentery characterised by polymorphous clinical picture in which many cases coursed in subclinical form could be reached only by using additional active methods for detecting the infection rate of the population. For this purpose the authors applied passive hemagglutination test which permitted to reveal the response of the organism to the antigenic stimulation in the course of two months after the sustained sickness. Over 12 000 persons were examined. According to the results of passive hemagglutination test seasonal activization of the epidemic process occurred one month earlier than it was revealed by recording of the incidence of the disease. The results of the mentioned test also showed infection rate of the population with Sonne dysentery to be as a rule greater than established by the official statistics.

The study of S. sonnei in phase I, irrespective of their virulence, has revealed the existence of at least 3 types of profiles of large plasmids: (I)A having a single plasmid with a molecular weight of about 120 MD; (I)B having, alongside plasmid pSS120, a plasmid with a molecular weight of about 60 MD; (I)C, represented only by vaccine strain 6S, having three plasmids with molecular weights of about 80, 60 and 37 MD. The plasmid profiles of rough S. sonnei in phase II are characterized by the absence of large plasmids with a molecular weight of 120-80 MD, typical of bacteria in phase I, and can be in their turn subdivided, in accordance with the type of the initial culture, into three subvariants (II)A, (II)B and (II)C. The plasmid profiles of rough S. sonnei (R-forms and phase II) completely coincide. The biosynthesis of the specific antigen of S. sonnei in phase I can be determined by smaller derivatives obtained from large plasmid pSS120 by deletion (e.g., by a plasmid with a molecular weight of about 80 MD, such as plasmid pSS80).

The paper presents the results of studying peculiarities of the Sh. sonnei of different biochemical types spread established by their typing scheme suggested by the authors earlier according to rhamnose, xylose and maltose. The epidemic process in dysentery both during the years of the rise and of the decline of its incidence at various territories of the countries proved to be maintained on account of circulation of Sh. sonnei of various biochemical types. The results of studying their dissociative and virulent properties confirmed the biological separation of individual biochemical types. An interrelationship between the character of the biochemical pattern of Shigellae sonnei at the individual territories and the persisting activity of different ways of dysentery transmission was determined. The results of studying the biochemical pattern could be used as an indicator of the degree of activity of individual ways of dysentery spread at various territories.

A series of Tn1 insertions in pSS120, the 120-megadalton form I plasmid of Shigella sonnei, were constructed by a Tn1-mediated conduction system previously described (H. Watanabe and A. Nakamura, Infect. Immun. 48:260-262, 1985, and screened for cell invasion in a tissue culture assay. The analysis of Tn1 insertion sites of seven noninvasive mutants suggested that four separate HindIII fragments were necessary for cell invasion. HindIII fragments including Tn1 of mutant plasmids were cloned into a vector plasmid, pACYC184. The DNA was used as a DNA probe to identify the corresponding, parental HindIII fragments. We identified one contiguous molecule of 2.6- and 4.1-kilobase pair (kb) HindIII fragments as being responsible for restoring cell invasiveness to the three mutant plasmids, pHW505, pHW510, and pHW511. Polypeptide analysis in minicells demonstrated that the contiguous HindIII fragments of 2.6 and 4.1 kb coded for at least four polypeptides, of 38, 41, 47, and 80 kilodaltons (kDa). A comparison of polypeptides synthesized by parental and mutant plasmids strongly suggested that the 38-kDa protein was essential for cell invasion. The 4.1-kb DNA which encoded the 38-kDa protein was conserved among plasmids of Shigella species and enteroinvasive Escherichia coli.

An attempt was made of using for the purpose of studying the virulence of Sh. sonnei of the method of determination of autoimmune plaque-forming cells in infected albino mice. By the mentioned method a total of 231 strains of Sh. sonnei of various biochemical types were examined. The virulence of 103 strains was checked in paralled by the keratoconjunctival test. It was shown that the method of determination of the autoimmune plaque-forming cells in the blood of infected mice could be used as a test for the virulence of Sh. sonnei, which was more sensitive than the keratoconjunctival test. Among the cultures studied 14.7% proved to be avirulent, 51.5%-of low virulence, 21.2%-moderately virulent, and 12.6%-highly virulent. Strains of the II biotype proved to be the most virulent, and strains of the III biotype-the least. The virulence of the cultures isolated from the patients with acute dysentery was more than double that of the cultures isolated from persons who came in contact with the patients.

The study of the structure and developmental dynamics of S. sonnei populations and the state of local immunity to this infection in children under school age has revealed that the interaction of the heterogeneous populations of the parasite and the host is the internal motive force of the self-regulating phasic development of the epidemic process. In the course of this process complementary changes in the virulence of the infective agent and in the immunoresistance of children are generated on the basis of negative feed-back.