Acid tolerance of Shigella sonnei and Shigella flexneri

AIMS: Determination of the behaviour of Shigella sonnei and Sh. flexneri under acid conditions. METHODS AND RESULTS: The growth and survival of Shigella spp. (9 isolates) in acidified Brain Heart Infusion (BHI) (pH 5.0-3.25 with pH intervals of 0.25) was determined after 6, 24 and 30 h incubation at 37 degrees C. Subsequently, survival of shigellae was studied in apple juice and tomato juice stored at 7 degrees C and 22 degrees C for up to 14 days and in strawberries and a fresh fruit salad, kept at 4 degrees C for 4 and 48 h. CONCLUSIONS: The minimum pH for growth in acidified BHI for Sh. flexneri and Sh. sonnei was, respectively, pH 4.75 and pH 4.50. Survival in fruit juices and fresh fruits depended upon their pH, the type of strain and the incubation temperature. Shigella spp. Survived for up to 14 days in tomato juice and apple juice stored at 7 degrees C. The shortest survival time (2-8 d) was observed in apple juice at 22 degrees C. Sh. sonnei but not Sh. flexneri was recovered after 48 h from strawberries and fruit salad kept at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Acid foods, especially if kept at refrigeration temperatures, support survival of Shigella spp. and may cause Shigella food poisoning.     

Gene cloning and characterization of alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei.

Alanine racemase genes (alr) from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei were cloned and expressed in Escherichia coli JM109. All genes encoded a polypeptide of 359 amino acids, and showed more than 99% sequence identities with each other. In particular, the S. dysenteriae alr was identical with the S. flexneri alr. Differences in the amino acid sequences between the four Shigella enzymes were only two residues: Gly138 in S. dysenteriae and S. flexneri (Glu138 in the other) and Ile225 in S. sonnei (Thr225 in the other). The S. boydii enzyme was identical with the E. coli K12 alr enzyme. Each Shigella alr enzyme purified to homogeneity has an apparent molecular mass about 43,000 by SDS-gel electrophoresis, and about 46,000 by gel filtration. However, all enzymes showed an apparent molecular mass about 60,000 by gel filtration in the presence of a substrate, 0.1 M l-alanine. These results suggest that the Shigella alr enzymes having an ordinary monomeric structure interact with other monomer in the presence of the substrate. The enzymes were almost identical in the enzymological properties, and showed lower catalytic activities (about 210 units/mg) than those of homodimeric alanine racemases reported.     

Use of coagglutination for serotyping Shigella dysenteriae and Shigella boydii

The coagglutination test was used to identify Shigella boydii and Shigella dysenteriae. A trial was carried out with 13 native rabbit antisera to S. boydii and 10 antisera to S. dysenteriae, as well as with coagglutinating reagents prepared from these antisera. The use of coagglutinating reagents was shown to ensure the complete specificity of the results, to prevent the adsorption of diagnostic antisera and to decrease their consumption 50 times. The importance of the coagglutination test for the identification of shigellae is discussed.     

Shigella and autophagy

Bacterial invasion of eukaryotic cells, and host recognition and elimination of the invading bacteria, determines the fate of bacterial infection. Once inside mammalian cells, many pathogenic bacteria enter the host cytosol to escape from the lytic compartment and gain a replicative niche. Recent studies indicate that autophagy also recognizes intracellular bacteria. Although autophagy is a conserved membrane trafficking pathway in eukaryotic cells that sequesters undesirable or recyclable cytoplasmic components or organelles and delivers them to lysosomes, autophagy has recently been described as playing a pivotal role as an intracellular surveillance system for recognition and eradication of the pathogens that have invaded the cytoplasm. Indeed, unless they are able to circumvent entrapping by autophagosomes, bacteria ultimately undergo degradation by delivery into autolysosomes. In this review we discuss recent discoveries regarding Shigella strategies for infecting mammalian cells, and then focus on recent studies of an elegant bacterial survival strategy against autophagic degradation.     

Survival of Shigella in Sewage: II. Effect of Glycerol on Shigella flexneri and Shigella Bacteriophage1

Glycerol (30%) inhibited or delayed the adsorption of Shigella bacteriophage on its host organism, S. flexneri II; glycerol also inhibited or delayed the burst of phage, whether or not adsorption was carried out in the presence of glycerol. Studies of the mechanisms of these effects showed that viscosity and osmotic shock probably were not responsible for either phenomenon. The inhibition of adsorption, however, was proportional to the concentration of glycerol, and appeared to be a function of the hydroxyl groups on the glycerol molecule. The inhibition of burst seemed to be related to the osmotic pressure outside the bacterial cells.     

An AFLP-based database of Shigella flexneri and Shigella sonnei isolates and its use for the identification of untypable Shigella strains

Amplified fragment length polymorphism (AFLP) can be used to assess the genetic diversity of closely related microbial genomes. In this study, the first of its kind for identification of Shigella, the high discriminatory power of AFLP has been used to determine the genetic relatedness of 230 isolates of Shigella flexneri and Shigella sonnei strains. An AFLP database was generated to demonstrate its utility in the discrimination of closely related strains. Based on AFLP, S. flexneri strains could be grouped into separate clusters according to their serotypes. Within each serotype, strains demonstrated 80–100% similarity indicating that identical strains and closely related strains could be distinguished by this technique. S. flexneri 6 formed a distinct cluster with 55% similarity to the rest of the S. flexneri strains showing significant divergence from the rest of the S. flexneri strains. Significantly, S. sonnei isolates formed a distinct group and showed approximately the same level of genetic linkage to S. flexneri as Escherichia coli strains. Untypable isolates that showed conflicting agglutination reactions with conventional typing sera were identifiable by AFLP. Thus AFLP can be used for genetic fingerprinting of Shigella strains and aid in the identification of variant untypable isolates.     

An AFLP-based database of Shigella flexneri and Shigella sonnei isolates and its use for the identification of untypable Shigella strains

Amplified fragment length polymorphism (AFLP) can be used to assess the genetic diversity of closely related microbial genomes. In this study, the first of its kind for identification of Shigella, the high discriminatory power of AFLP has been used to determine the genetic relatedness of 230 isolates of Shigella flexneri and Shigella sonnei strains. An AFLP database was generated to demonstrate its utility in the discrimination of closely related strains. Based on AFLP, S. flexneri strains could be grouped into separate clusters according to their serotypes. Within each serotype, strains demonstrated 80-100% similarity indicating that identical strains and closely related strains could be distinguished by this technique. S. flexneri 6 formed a distinct cluster with 55% similarity to the rest of the S. flexneri strains showing significant divergence from the rest of the S. flexneri strains. Significantly, S. sonnei isolates formed a distinct group and showed approximately the same level of genetic linkage to S. flexneri as Escherichia coli strains. Untypable isolates that showed conflicting agglutination reactions with conventional typing sera were identifiable by AFLP. Thus AFLP can be used for genetic fingerprinting of Shigella strains and aid in the identification of variant untypable isolates.     

Characterization of cryptic flagellin genes in Shigella boydii and Shigella dysenteriae.

Flagellin (fliC) genes of 12 Shigella boydii and five Shigella dysenteriae strains were characterized. Though these strains are nonmotile, the cryptic fliCSB gene, cloned from S. boydii strain C3, is functional for expression of flagellin. It consists of 1,704 bp, and encodes 568 amino acid residues (57,918 Da). The fliCSD gene from S. dysenteriae strain 16 consists of 1,650 bp encoding 549 amino acid residues (57,591 Da) and contains an IS1 element inserted in its 3' end. The two genes are composed of the 5'-constant, central variable and 3'-constant sequences, like other known fliC genes. The two genes share high homology in nucleotide and amino acid sequences with each other and also with the Escherichia coli fliCE gene, indicating that both genes are closely related to the fliCE gene. Comparison of the central variable sequences of six different fliC genes showed that the fliCSB and fliCSD genes share low homology in amino acid sequence with the other fliC genes, suggesting that they encode antigenic determinants intrinsic to respective subgroups. However, Southern blotting using as probes the central variable sequences of several fliC genes showed that four of 12 S. boydii strains have a fliC gene similar to that of Shigella flexneri, and that among five fliC genes from S. dysenteriae strains, one is similar to that of S. flexneri, two are similar to that of S. boydii, and only one is unique to S. dysenteriae. Some of these variant alleles were verified by immunoblotting with flagellins produced from cloned fliC genes. The presence of variant fliC alleles in S. boydii and S. dysenteriae indicates that subdivision into subgroups does not reflect the ancestral flagella H antigenic relationships. These data will be useful in considering the evolutionary divergence of the Shigella spp..     

Intracellular survival of Shigella

Bacterial invasion of eukaryotic cells and host recognition and killing of the invading bacteria are a key issue in determining the fate of bacterial infection. Once inside host cells, pathogenic bacteria often modify the phagosomal compartment or enter the host cytosol to escape from the lytic compartment and gain a replicative niche. Cytosolic invaders, however, are monitored by host innate immune systems, such as mediated by Nod/CARD family proteins, which induce inflammatory responses via activation of NF-kappaB. Furthermore, recent studies indicate that autophagy, a major cytoplasmic degradation system that eliminates cytosolic protein and organelles, also recognizes invading bacteria. Indeed, unless they are able to circumvent entrapping by autophagic membranes, bacteria targeted by autophagy ultimately undergo degradation by delivery into autolysosomes. In this article, we review recent advances in understanding of Shigella strategies to infect epithelial cells, and then focus on recent studies of an intriguing bacterial survival strategy against autophagic degradation.     

Lipopolysaccharides of Shigella sonnei

Immunobiological properties of native lipopolysaccharides (LPS) from virulent and avirulent strains of Shigella sonnei bacteria (LPS-V and LPS-A, respectively) were studied. In avirulent bacteria, LPS-V induced immunosuppressive activity specific of the virulent strain. LPS of the avirulent strain, whereas LPS-A lacked this property. Native LPS-V with immunosuppressive activity were isolated from the virulent strain by and immune affinity method. Treatment of LPS-V with phenol or TCA abolished its activity and converted it into the LPS-A form. The data showed that LPS-A can be converted back to the LPS-V form by redox treatment. This approach seems to be promising for activating LPS extracted from cells with TCA or a water-phenol mixture.