Epidemias por salmonella y por shigella, importancia de la vigilancia epidemiologíca y control de la resistencia a los antibióticos / Epidemics by salmonella and shigella, importance of epidemiologic vigilance and control of the antibiotics resistence

El descubrimiento de los antibióticos y su aplicación clínica ha permitido el tratamiento y control de padecimientos infecciosos. Por otra parte, se ha descubierto que algunas enterobacterias desarrollan resistencia a las drogas antimocrobianas causada, principalmente por la presencia de elementos extracromosómicos denominados plasmidos. De entre estos microorganismos se han estudiado la salmonella y shigella con la finalidad de determinar las características de las mismas, los antibióticos a que se resisten y con cuales pueden ser tratados los padecimientos provocados por las mismas. La presencia de epidemias y las endemias en regiones de México así como en otras regiones del mundo, y consecuentemente el alto índice de morbilidad han inducido a los especialistas a investigar los procesos infecciosos provocados por shigella y la salmonella, se ha logrado establecer una clasificación de las mismas así como la sintomatología que producen las mismas. Para establecer un tratamiento determinado, se aisló al agente infeccioso, se le sometió a estudios para poder establecer a que drogas eran resistentes y a cuales no. De este modo se ha podido descrubir que algunos tipos de estas bacterias son resistentes, por ejemplo, al clorafenicol, la tetraciclina, la estreptomicina y a la ampicilina, en tanto que otras son suceptibles a los mismos, así mismo se ha observado que la región guarda cierta relación con el desarrollo de la resistencia

Cell surface proteins from Shigella dysenteriae type 1.

A simple extraction procedure was used for preparing cell surface proteins (CSPs) from Shigella dysenteriae type 1. The preparations obtained using either buffer or water extractions were free from lipopolysaccharide (LPS), as well as cytoplasmic and periplasmic proteins. By SDS-PAGE, about 25 polypeptides were detected, and Western-blot analysis recognised 15 polypeptide antigens. When analysed by crossed immunoelectrophoresis, using anti-Shigella dysenteriae type 1 rabbit sera, 18 antigenic bands were identified. Proteins obtained by this method were found to be highly immunogenic in rabbits. The cell-surface proteins were compared to outer membrane proteins (OMPs) obtained from the S. dysenteriae type 1 strain by a standard procedure involving lysozyme-EDTA extraction, sucrose density centrifugation, and detergent treatment. They were found to contain periplasmic, cytoplasmic, and lipopolysaccharide contaminants. Thus, the procedure described here offers a quick and simple alternative for obtaining relatively pure cell surface proteins from Shigella dysenteriae type 1. This method will be useful when immunogenically active proteins free from other cellular components are required for studies.

Immunodetection of lipopolysaccharide in macrophages during the processing of non invasive Shigella dysenteriae.

The location of lipopolysaccharide (LPS) was studied by immunofluorescence and immunoelectron microscopy in macrophages infected with a non-invasive Shigella dysenteriae 1 strain. Bacterial degradation began only 3 h after the end of infection. The first visible sign of degradation was detected by immunogold labelling at the level of LPS which detached from the bacterial surface and was transferred to the perinuclear lysosomes. After a few hours, it was found in small vesicles spread over the whole macrophage cytoplasm in which it remained visible for 72 h. These vesicles seemed to belong to a compartment in which slowly or non-degradable compounds are stored. LPS separation from the bacterial surface was immediately followed by the degradation of the intrabacterial constituents. The long lag period observed before initiation of bacterial degradation was not due to a lack of phagosome acidification, since DAMP, a lysosomotropic drug was found in all phagosomes at the end of the ingestion period. The frequency of phagosome-lysosome fusion was 30% for S dysenteriae and 72% for B subtilis used as a reference of high fusion frequency. The low frequency of fusion of S dysenteriae may play an important role in the survival of the virulent strains in macrophage by providing bacteria enough time to lyse the phagosome membrane before lysosome fusion occurs.

Isolation and characterization of functional Shiga toxin subunits and renatured holotoxin.

Shiga toxin is a protein toxin produced by Shigella dysenteriae type I strains. In this report we present a procedure for the separation of functionally intact toxin A and B chains and for their reconstitution to form biologically active molecules. In agreement with the findings of others, the isolated A chain was shown to be a potent in vitro inhibitor of eukaryotic protein synthesis. The isolated B chain bound to HeLa cells and competitively inhibited the binding and cytotoxic activity of holotoxin. These findings show that the functional role of the B chain is to recognize cell surface functional receptors. By labelling the B subunit alone, prior to renaturation of holotoxin, the polypeptide chains were shown to associate noncovalently with a stoichiometry of one A chain and five B chains.

Interaction of Shigella toxin with globotriaosyl ceramide receptor-containing membranes: a fluorescence study.

The interaction of the B-subunit of Shigella toxin with a globotriaosyl ceramide receptor incorporated into phosphatidylcholine vesicles was studied by fluorescence spectroscopy. From the position of the maximum in the emission spectrum and the accessibility to acrylamide quenching, it is concluded that a single tryptophan of a free B-chain is located in a highly polar environment, most likely on the surface of the folded polypeptide chain. Binding of B-subunits to the membrane-associated globotriaosyl ceramide results in a strong enhancement of fluorescence intensity and a small blue-shift of the emission maximum; these effects suggest a conformational change in the protein which provides a new environment to a tryptophan residue. However, the polarity of this new environment is still relatively high--as indicated by the position of the emission maximum at 344 nm--and suggests that the receptor-bound B-chain remains largely on the membrane surface, without penetrating the hydrophobic interior of a lipid bilayer. On the other hand, the A-chains are shown to interact directly with the receptor-free lipid bilayers; this nonspecific interaction may play a role in the mechanism by which A-subunit traverses the membrane of a target cell.

[Differentiation of virulent strains of Shigella and entero-invasive Escherichia from their avirulent variants using modified indirect immunoenzyme analysis]. / Differentsiatsiia virulentnykh shtammov shigell i énteroinvazivnykh ésherikhii ot ikh avirulentnykh variantov pri pomoshchi modifitsirovannogo nepriamogo immunofermentnogo analiza.

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.

The stability of O-antigen plasmid is determined by a chromosomal region of Shigella dysenteriae 1.

It is well established that plasmids are involved in the expression of lipopolysaccharide in certain species of Shigella. In Shigella sonnei, both the biosynthesis of oligosaccharide side chains (O antigen), and cell invasiveness are controlled exclusively by a 120 megadalton (MDa) plasmid. In Shigella dysenteriae 1, a 10 kilobase (kb) plasmid is required for O-antigen production. Shigella dysenteriae 1 strains devoid of this plasmid lose the ability to synthesize O antigen. Interestingly, this 10-kb plasmid is not stably maintained in Escherichia coli K-12 strains, where it is lost spontaneously at a high frequency. Our genetic analyses of Shigella dysenteriae 1 strain IDBM11 and its derivatives indicate that the stability of this plasmid is associated with the histidine region of the chromosome which is unique to Shigella dysenteriae. Furthermore, the 10-kb plasmid is stably maintained in wild-type IDBM11 with an intact histidine locus. However, this plasmid is not stable in IDBM11 derivatives (e.g., IDBM11-1 and IDBM11-2), in which the his locus has been substituted with the histidine region of an E. coli K-12 chromosome. The S. dysenteriae IDBM11 strain, and its derivatives (lacking a 10-kb plasmid), displayed an invasive property as demonstrated by their internalization by HeLa cells in an in vitro assay. Thus the 10-kb plasmid of Shigella dysenteriae 1 is required for O-antigen synthesis but not for cell invasion.

Characterization of purified Shiga toxin from Shigella dysenteriae 1.

Shiga toxin was purified from the culture supernatant of Shigella dysenteriae 1 by ammonium sulfate fractionation, DEAE-cellulose column chromatography and repeated chromatofocusing column chromatography. About 1.6 mg of purified Shiga toxin was obtained from 15 liters of culture with a yield of about 27%. The molecular weight of purified Shiga toxin was estimated to be 62,000. The toxin consisted of A and B subunits with molecular weights of about 30,000 and 5,000-6,000, respectively. The isoelectric point of purified Shiga toxin was 7.0. Purified Shiga toxin showed the following biological activities: lethal toxicity to mice when injected intraperitoneally with an LD50 of 28 ng per mouse; cytotoxicity to Vero cells, killing about 50% of the cells at 1 pg and all of the cells at 10 pg; and fluid accumulation in rabbit ileal loops at a concentration of more than 1 microgram.

Immunoblotting procedure for the analysis of electrophoretically-fractionated bacterial lipopolysaccharide.

A procedure is described for the efficient transfer of fractionated bacterial lipopolysaccharide (LPS) from SDS-polyacrylamide gels to nitrocellulose filters, and its subsequent display by a peroxidase-linked antibody. The method is sensitive, and reveals and resolves high molecular weight LPS molecules having side chain lengths of up to and greater than 30 repeat units. It is useful for the rapid analysis of LPS in bacterial outer membrane preparations.

Purified Shigella enterotoxin does not alter intestinal motility.

A purified Shigella enterotoxin (pST) and a cell-free lysate with pST removed (CFL-pST) from the whole-cell lysate of Shigella dysenteriae 60 R were used to study their effect on the myoelectric activity and mucosal integrity of rabbit ileal segments. We have previously defined two myoelectric patterns: the migrating action potential complex and repetitive bursts of action potentials that occur in response to certain bacteria and their enterotoxins. The in vivo model consisted of isolated ileal segments in male New Zealand White rabbits. The segments were infused with sterile saline (1 ml/h), pST (2.4-micrograms injection), or CFL-pST (1 ml/h). Myoelectric activity in the segments exposed to pST was similar to that with the saline infusion, but CFL-pST induced significant alterations in myoelectric activity in the form of repetitive bursts of action potentials. The mucosa of the segments exposed to pST showed only mild inflammatory changes. In contrast, CFL-pST caused moderate to severe inflammatory changes with enterocyte necrosis. These studies show that pST, a known enterotoxin, did not alter myoelectric activity and had no significant effect on the integrity of ileal mucosa, as determined by light microscopy. CFL-pST caused both inflammation and tissue necrosis with significant alterations in motor activity. These studies suggest that S. dysenteriae 60 R produces a substance or substances other than pST that cause florid in vivo cytotoxicity and alter myoelectric activity.

Isolation and characterization of monoclonal antibodies to Shiga toxin.

Hybridoma cell lines which produce monoclonal antibodies to Shiga toxin from Shigella dysenteriae 1 were prepared. The monoclonal antibodies were all of the immunoglobulin G1 isotype and differed in their ability to neutralize cytotoxicity and to bind to Shiga toxin in a solid-phase radioimmunoassay. When used for immunoblot analysis, these antibodies were able to identify specifically both nicked and unnicked Shiga toxin in crude lysates of S. dysenteriae.

Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli.

A toxin from an enteropathogenic strain of Escherichia coli (E. coli H30) was purified to apparent homogeneity from cell lysates. The steps used to isolate the E. coli H30 toxin included French pressure-cell disruption of bacteria grown in iron-depleted media. Affi-Gel Blue chromatography, chromatofocusing, and anti-Shiga toxin affinity chromatography. The mobilities of the subunits of radioiodinated E. coli H30 toxin and Shiga toxin observed after the two toxins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis were identical. In the absence of 2-mercaptoethanol, a narrow band was seen at Mr 31,500 (+/- 1,000), and a wide heavy band was observed between Mr 4,000 and 15,000. In the presence of 2-mercaptoethanol, bands were seen at Mr 31,500 (+/- 1,000), 27,000, and 4,000 to 15,000. Other similarities between purified E. coli H30 and Shiga 60R toxins included identical isoelectric points (7.03 +/- 0.02); comparable biological activities, i.e., cytotoxicity, lethality for mice, and enterotoxicity; and the same relative heat stabilities (up to 65 degrees C for 30 min). Nevertheless, the two toxins had apparently different molecular weights as determined by sucrose gradient analysis, by gel filtration, and by cross-linking experiments with dimethyl suberimidate. The Mr of native E. coli H30 toxin estimated from cross-linking studies was 48,000, whereas the estimated Mr of Shiga 60R toxin was 58,000. These results suggest that like the cholera-E. coli-heat-labile toxin family, a family of Shiga-like toxins exists.

Characteristics of lipid A-protein complex from endotoxin of Shigella dysenteriae type 1 (S and R strains).

Mild acetic acid hydrolysis of endotoxin (lipopolysaccharide-protein complex) of Shigella dysenteriae type 1 (S and R forms) yielded a lipid A-protein complex that consisted of amino acids, fatty acids, and sugar and, in terms of chemical composition, displayed no marked differences between the S and R forms. Its protein portion (53 to 56%) consisted of at least 16 amino acids. In the fatty acid portion (14 to 18%), myristic, 3-hydroxymyristic, palmitic, and stearic acids accounted for 50%. The sugar portion (10 to 12%) consisted solely of glucosamine. The remainder was unidentified substances, most of which contained phosphorus. Lipid A-protein complexes derived from both S and R forms were not toxic for mice in doses up to 1,000 microgram/mouse, but their Linulus test activity had increased considerably as compared with the starting lipopolysaccharide-protein complex material: from 10(-6) to 10(-10--10(-12) mg/ml. The lipid A-protein complexes were readily soluble in a water solution of triethylamine, in dimethyl sulfoxide, and in pyridine.

Characterization of an R-plasmid associated with ampicillin resistance in Shigella dysenteriae type 1 isolated from epidemics.

Ampicillin-resistant strains of Shigella dysenteriae type 1 isolated in epidemics in Mexico, Central America, and Bangla Desh were examined for the presence of plasmid deoxyribonucleic acid (DNA) by gel electrophoresis. All strains contained a heterogeneous population of plasmids. Transfer experiments to Escherichia coli K-12 indicated that the ampicillin resistance determinant (Ap(r)) was located on a 5.5-megadalton (Mdal) plasmid identical in all Shiga strains examined, as judged by DNA hybridization and by its molecular properties. This 5.5-Mdal plasmid contained the ampicillin transposon (TnA) sequences. There was not a high degree of homology between the Shiga Ap(r) plasmid DNA and DNA obtained from Ap(r)Salmonella typhi strains isolated from typhoid epidemics in Mexico, previous to the dysentery outbreaks. Although low, the degree of reassociation observed indicated that probably part of the TnA sequence was present in S. typhi DNA. The DNA hybridization experiments showed, in addition, that there was a high degree of homology among Ap(r) plasmids isolated from different enterobacteria, and this identity was confirmed by restriction endonuclease activity. These results together with their similarities in molecular and replicative properties indicate that the Ap(r) plasmids, as was suggested for the Sm(r) Su(r) plasmids, possibly evolved once and then epidemiologically spread in the Enterobacteriaceae.

New sugars from antigenic lipopolysaccharides of bacteria: identification and synthesis of 3-O-[(R)-1-carboxyethyl]-L-rhamnose, an acidic component of Shigella dysenteriae type 5 lipopolysaccharide.

A new acidic sugar, 3-O-[(R)-1-carboxyethyl]-L-rhamnose (1), has been identified as a constituent of the O-antigenic lipopolysaccharide of Sh. dysenteriae type 5. The structure of 1 has been established by physico-chemical methods and by synthesis. Alkylation of methyl 2,5-di-O-benzyl-alpha-L-rhamnofuranoside (6) with (S)- or (R)-2-chloropropionic acids, followed by removal of the protecting groups, afforded 3-O-[(R)-1-carboxyethyl]-L-rhamnose (9) and 3-O-[(S)-1-carboxyethyl]-L-rhamnose (10), respectively. The properties of 1 coincide with those of 9.

Somatic antigens of shigella. Structural investigation on the O-specific polysaccharide chain of Shigella dysenteriae type 1 lipopolysaccharide.

The O-specific polysaccharide obtained from the lipopolysaccharide of Shigella dysenteriae type 1 (Shigella shiga) by mild acid hydrolysis followed by fractionation on Sephadex G-50 was found to be identical to that desribed by Morgan's group and was composed of L-rhamnose, D-galactose and N-acetyl-D-glycosamine in a ratio 2:1:1. On the basis of methylation analysis data the polysaccharide was proved to be a linear chain of monosaccharide residues in pyranose forms substituted at position 3, except for that of galactose substituted at position 2. Selective cleavage, based on the N-deacetylation reaction of the polymer, together with determination of linkage configurations by chromic anhydride oxidation showed that the O-specific polysaccharide is built up of repeating tetrasaccharide units whose proposed structure is given below -3)-alpha-L-Rhap (1-3)-alpha-L-Rhap(1-2)-alpha-D-Galp(1-3)-alphapD-GlcNAcp(1- where RHAP = rhamnopyranose, Galp = galactopyranose, and GlcNAcp = N-acetyl-glucosamine. The present findings confirmed the considerations of Heidelberger on the substitution patterns of L-rhamnose and D-galactose residues from the results of serological studies.

Selective cleavage of glycosidic linkages: studies with the polysaccharide component of Shigella dysenteriae type 6 lipopolysaccharide.

The polysaccharide component obtained from the lipopolysaccharide of Shigella dysenteriae type 6 was subjected to milk hydrolysis with acid, and the products were fractionated on Sephadex G-50. An acidic hexosaminoglycan and a core oligosaccharide fraction were obtained, the former containing D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose (in the ratios 1:1:1), and an unidentified acidic component (X). The hexosaminoglycan was N-deacetylated and then hydrolysed and deaminated to give 3-O-(2-amino-2-deoxy-beta-D-galactopyranosyl)-D-galactose (1), identified as the N-acetyl derivative (2), and 2,5-anhydro-3-O-(6-O-alpha-D-galactopyranosyl-alpha-D-glucopyranosyl)talitol (3). On the basis of the structure of 2 and the methylation-analysis data for the polysaccharide and 3, together with that for the determination of linkage configurations by chromic anhydride oxidation, the hexosaminoglycan is considered to have the repeating structure (see article).

Selective cleavage of glycosidic linkages: studies with the O-specific polysaccharide from Shigella dysenteriae type 3.

Treatment of the O-specific polysaccharide from Shigella dysenteriae Type 3 with hydrazine in the presence of hydrazine sulphate resulted in quantitative N-deacetylation with the formation of a modified polysaccharide containing free amino groups. Oxidation of the modified polysaccharide with periodate did not destroy the 2-amino-2-deoxygalactose residues, thus indicating that they were substituted at position 3. Acid hydrolysis of the modified polysaccharide afforded 3-O-(2-amino-2-deoxy-beta-D-galactopyranosyl)-D-galactose, which was identified as the N-acetyl derivative. Deamination of the modified polysaccharide with nitrous acid cleaved the 2-amino-2-deoxy-D-galactopyranosyl linkages to give a pentasaccharide as the major product, which appeared to be the modified chemical repeating unit of the O-specific polysaccharide.