Differentiation of Shigella by esterase electrophoretic polymorphism

The electrophoretic mobilities of four esterases (A, B, C, and I) of 182 strains of Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei were compared to those of 636 strains of Escherichia coli from various origins, including the Alkalescens Dispar group and enteroinvasive strains. Discriminant analysis of the distribution of esterases among the strains revealed that Shigella could be distinguished from E. coli by differences in the distribution of allozymes of esterases C and I. Principal components analysis distinguished four major clusters of Shigella strains corresponding to the following: S. dysenteriae serotype 1; S. flexneri serotypes 1 to 5; S. flexneri serotype 6 and S. boydii serotypes 2 and 4; and S. sonnei. The last three were characterized by distinct electrophoretic variants of carboxylesterase B, as judged by the two-dimensional electrophoretic profile and titration curves. The distinct esterase pattern obtained for the strains of S. boydii serotype 13 substantiates the view that this serotype may constitute a new species.     

Molecular cloning and genetic analysis of the rfb region from Shigella flexneri type 6 in Escherichia coli K-12

The rfb gene cluster which determines the biosynthesis of the Shigella flexneri serotype 6 O-antigen specificity has been cloned in pHC79, generating plasmids pPM3115 and pPM3116. These plasmids mediate expression, in Escherichia coli K-12, of lipopolysaccharides (LPS) immunologically similar to the S. flexneri type 6 LPS as judged by SDS-PAGE and Western-immunoblot analysis using S. flexneri type 6 specific antisera. Thus, unlike other S. flexneri serotypes, no additional loci are required for serotype specificity. This expression is independent of E. coli K-12 rfb genes. Southern-hybridization analysis using the 16.2-kb BglII probe from S. flexneri type 6 rfb region detected very little sequence homology in S. flexneri serotypes 1-5, however, some homology was detected with E. coli O2 and O18, but not in E. coli 0101 strains, Salmonella and Vibrio cholerae.     

The factor C3 conversion in human complement by smooth Shigella flexneri lipopolysaccharides

Shigella flexneri rods play an important role in human intestinal infections. In the presented studies we have shown that O-acetyl and glucose residues, substituted in main GalNAc-Rha chains of lipopolysaccharide (LPS) are important for the bactericidal effect of human serum. By dot-blot, immunoblotting and ELISA with immobilized LPS we have shown correlation of C3 fragments deposition and serum resistance. LPSs isolated from a serum-sensitive strain deposited more C3 fragments than LPSs from serum-resistant Shigella flexneri strains.     

Sensitivity of Shigella strains to the bactericidal activity of human serum. II. Shigella flexneri 3 a is killed by complement activated via the classical pathway, with participation of lysozyme.

Normal human serum is bactericidal for all studied strains (15) of Shigella flexneri serotype 3a. The activity of the serum was similar irrespective of the invasiveness of the bacteria or its lack. The studied bacteria were susceptible to a single mechanism of bactericidal activity involving complement activated via the classical pathway, accompanied by the action of lysozyme.     

Differentiation of Shigella strains by plasmid profile analysis, serotyping and phage typing

Ninety-one Shigella flexneri and 29 Shigella sonnei strains isolated during 1994 from sporadic cases of shigellosis and healthy carriers were analyzed for plasmid profile in order to compare the discriminating ability of this method with that of serotyping and phage typing. Our study revealed 10 plasmid profiles (PP) among S. sonnei strains. A total of 26 out of 29 (89%) S. sonnei isolates could be placed into two phage types (type 1 and 20) comprising four PP for phage type 1 and seven PP for type 20, respectively. Twenty-three different PP were identified among S. flexneri strains. Each serotype was associated with a specific predominant plasmid profile, except serotype 2a. This serotype, the most frequently isolated in Romania, was still rather homogeneous: 33 out of 39 isolates belonged to phage type 125, 27 of which could be placed into two related PP (F10 and F17). Comparison of plasmid patterns of epidemiologically independent S. flexneri serotype 2a isolates with those exhibited by 45 serotype 2a isolates associated to six independent outbreaks revealed the same homogeneity. Thirty-eight strains, representing 4 of 6 outbreaks, had F10 and F17 plasmid patterns. The discrimination indices (D) for plasmid profile analysis alone (D = 0.890) and for the combination of serotyping and phage typing (D = 0.841) indicate that both typing systems have a nearly similar ability of discriminating among S. flexneri strains. By combining the results of the three typing methods, a total of 42 types are distinguished and the D value is 0.942. Our data suggest that plasmid profile analysis can complement phenotyping methods resulting in a degree of discrimination that cannot be achieved by either system alone.     

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.     

New animal model of shigellosis in the Guinea pig: its usefulness for protective efficacy studies

It has been difficult to evaluate the protective efficacy of vaccine candidates against shigellosis, a major form of bacillary dysentery caused by Shigella spp. infection, because of the lack of suitable animal models. To develop a proper animal model representing human bacillary dysentery, guinea pigs were challenged with virulent Shigella flexneri serotype 2a (strains 2457T or YSH6000) or S. flexneri 5a (strain M90T) by the intrarectal (i.r.) route. Interestingly, all guinea pigs administered these Shigella strains developed severe and acute rectocolitis. They lost approximately 20% of their body weight and developed tenesmus by 24 h after Shigella infection. Shigella invasion and colonization of the distal colon were seen at 24 h but disappeared by 48 h following i.r. infection. Histopathological approaches demonstrated significant damage and destruction of mucosal and submucosal layers, thickened intestinal wall, edema, erosion, infiltration of neutrophils, and depletion of goblet cells in the distal colon. Furthermore, robust expression of IL-8, IL-1beta, and inducible NO synthase mRNA was detected in the colon from 6 to 24 h following Shigella infection. Most importantly, in our new shigellosis model, guinea pigs vaccinated with an attenuated S. flexneri 2a SC602 strain possessing high levels of mucosal IgA Abs showed milder symptoms of bacillary dysentery than did animals receiving PBS alone after Shigella infection. In the guinea pig, administration of Shigella by i.r. route induces acute inflammation, making this animal model useful for assessing the protective efficacy of Shigella vaccine candidates.     

鲍氏志架氏菌的一个新血清型

Two strains which belong to the same serotype of Shigella were isolated from the bloody-pus stool of two patients (in 1986) and is reported in this paper. The results were identical both showing agglutination in low titer with serotype 8 of S. dysenteriae and serotype 4 of S. boydii when the two strains were checked well with all kinds of diagnostic antisera and vice versa, ie the antisera produced by the two strains were also checked well with sera prepared with the representative strains of all Shigella spp. No cross agglutination with O6, O7, and O150 of E. coli were found. Consequently, It appears to be a new serotype of Shigella. These two strains possess the ability of causing keratitis in guinea-pigs as well as invading epithelial cells, the DNA of both strains in agarose-electrophoresis showed a large plasmid, indicating that they are virulent strains possessing invasive ability. It was concluded that these two strains belonged to Shigella boydii as they fermented mannitol and non-related antigenically with Shigella flexneri. Since serotype 1-18 of S. boydii have been reported recently, we propose that this new serotype should be serotype 19 of Shigella boydii.     

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli ; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli , had lipopolysaccharide profiles quite distinct from the respective strain of E. coli . It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.     

Identification of a putative pathogenicity island in Shigella flexneri using subtractive hybridisation of the S. flexneri and Escherichia coli genomes

The genetic differences between the human pathogen, Shigella flexneri, and the non-pathogenic Escherichia coli were investigated in an attempt to identify pathogenicity islands (PAIs) in the S. flexneri genome. Genomic subtraction identified a large unique region of DNA which was present in S. flexneri serotype 2a but absent from E. coli K-12. This 42-kb DNA segment was localised to the S. flexneri chromosome and was found to contain a number of elements often associated with PAIs including: insertion sequence elements, bacteriophage genes, and a previously identified Shigella virulence gene (criR). These findings indicate that this region may form a new PAI in the S. flexneri genome.     

Bacteria-infected fibroblasts have enhanced susceptibility to the cytotoxic action of tumor necrosis factor.

The susceptibility of bacteria-infected fibroblasts to the cytotoxic action of tumor necrosis factor was investigated. L cells infected with Shigella flexneri, Salmonella typhimurium, or Listeria monocytogenes, had an enhanced susceptibility to the cytotoxic activity of TNF-alpha. This enhanced susceptibility was dependent upon the challenge dose of bacteria, the concentration of TNF, and upon the exposure time of bacteria-infected cells to TNF. L cells infected with S. flexneri were susceptible to the cytotoxic action of TNF at 2 to 6 h after bacterial infection. In contrast, L cells infected with S. typhimurium or L. monocytogenes did not show enhanced susceptibility to TNF until 14 h postbacterial infection and exposure to TNF. Enhanced susceptibility to TNF was dependent on bacterial invasion because fibroblasts pretreated with a noninvasive isogenic variant of S. flexneri, UV-treated invasive bacteria, bacterial cultural supernatant, or bacteria LPS were no more susceptible to TNF than untreated cells. Enhanced susceptibility to TNF by bacteria-infected cells was not unique to L cells. Mouse embryo fibroblasts and HeLa cells also showed similar reactivities after bacteria infection. Bacteria-infected cells were greatly suppressed in host cell protein synthesis that may play an important role in their enhanced susceptibility to TNF. These results suggest that an important role of TNF in host defense against bacterial infections is its cytotoxic activity against bacteria-infected cells.     

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.     

Effects of bacterial lipopolysaccharide on the binding of lymphocytes to endothelial cell monolayers

Preincubation of human umbilical vein endothelial cell (EC) monolayers with 1 ng to 10 micrograms/ml lipopolysaccharide (LPS) increased the binding of T lymphocytes to EC. The effect was maximal at LPS concentrations of 0.1 to 10 micrograms/ml, and occurred with LPS derived from Escherichia coli (serotypes 0111:B4 and 0127:B8), Shigella flexneri (serotype 2a), Serratia marcescens (serotype 0:3), and Yersinia entercolitica (serotype 0:3). The increased binding appeared to be mediated primarily through an action on EC; preincubation of T cells rather than EC with LPS did not lead to enhanced binding. The onset of enhanced binding was very rapid, being observed after 2 to 3 min of preincubation and becoming maximal after 1 hr. EC were unresponsive to LPS after fixation with 2% paraformaldehyde-L-lysine-periodate and also when the LPS was incubated with EC at 4 degrees C. Enhanced binding was seen with lipid A and with LPS from Salmonella minnesota Re 595 (mainly lipid A) and was abolished by conjugation with polymyxin B. The observed increase in the binding of lymphocytes to EC exposed to LPS suggests that the lymphocytopenia induced by endotoxemia may result from augmentation of the adherence of lymphocytes to altered endothelium.     

Identification of an Escherichia coli gene homologous to virR, a regulator of Shigella virulence.

Virulence in Shigella spp., as well as in strains of enteroinvasive Escherichia coli, is regulated by growth temperature. Previously, virR had been identified as the gene controlling the temperature-regulated expression of Shigella virulence. Since Shigella spp. and E. coli are also known to share greater than 90% DNA sequence homology, we sought to determine if nonpathogenic E. coli K-12 C600 contains a gene homologous to the Shigella flexneri 2a gene virR. Through the use of transduction and molecular cloning of strain C600 chromosomal DNA we have shown that E. coli K-12 does indeed contain a gene functionally homologous to the virR of S. flexneri.     

Shigella flexneri regulates tight junction-associated proteins in human intestinal epithelial cells

Shigella spp. are a group of Gram-negative enteric bacilli that cause acute dysentery in humans. We demonstrate that Shigella flexneri has evolved the ability to regulate functional components of tight junctions after interaction at the apical and basolateral pole of model intestinal epithelia. In the regulation of tight junctional protein assemblies, S. flexneri can engage serotype-specific mechanisms, which targets not only expression, but also cellular distribution and membrane association of components of tight junctions. Distinct mechanisms resulting in the regulation of tight junction-associated proteins are initiated after either apical or basolateral interactions. S. flexneri serotype 2a has the ability to remove claudin-1 from Triton X-insoluble protein fractions upon apical exposure to T-84 cell monolayers. S. flexneri serotype 2a and 5, but not the non-invasive Escherichia coli strain F-18, share the ability to regulate expression of ZO-1, ZO-2, E-cadherin and to dephosphorylate occludin. The disruption of tight junctions is dependent on direct interaction of living Shigella with intestinal epithelial cells and is supported by heat-stable secreted bacterial products. Intestinal epithelial cells have the ability to compensate in part for S. flexneri induced regulation of tight junction-associated proteins.     

Frequency of serovars and antimicrobial resistance in Shigella spp. from Brazil

A total of 296 Shigella spp. were received from State Public Health Laboratories, during the period from 1999 to 2004, by National Reference Laboratory for Cholera and Enteric Diseases (NRLCED)--IOC/Fiocruz, Rio de Janeiro, Brazil. The frequency of Shigella spp. was: S. flexneri (52.7%), S. sonnei (44.2%), S. boydii (2.3%), and S. dysenteriae (0.6%). The most frequent S. flexneri serovars were 2a and 1b. The highest incidence rates of Shigella isolation were observed in the Southeast (39%) and Northeast (34%) regions and the lowest rate in the South (3%) of Brazil. Strains were further analyzed for antimicrobial susceptibility by disk diffusion method as part of a surveillance program on antimicrobial resistance. The highest rates of antimicrobial resistance were to trimethoprim-sulfamethozaxole (90%), tetracycline (88%), ampicillin (56%), and chloramphenicol (35%). The patterns of antimicrobial resistance among Shigella isolates pose a major difficulty in the determination of an appropriate drug for shigellosis treatment. Continuous monitoring of antimicrobial susceptibilities of Shigella spp. through a surveillance system is thus essential for effective therapy and control measures against shigellosis.     

Full structural characterization of Shigella flexneri M90T serotype 5 wild-type R-LPS and its delta galU mutant: glycine residue location in the inner core of the lipopolysaccharide

Shigella flexneri is a gram-negative bacterium responsible for serious enteric infections that occur mainly in the terminal ileum and colon. High interest in Shigella, as a human pathogen, is driven by its antibiotic resistance and the necessity to develop a vaccine against its infections. Vaccines of the last generation use carbohydrate moieties of the lipopolysaccharide as probable candidates. For this reason, the primary structure of the core oligosaccharide from the R-LPS produced by S. flexneri M90T serotype 5 using chemical analysis, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MALDI), is herein reported. This is the first time that the core oligosaccharide primary structure by S. flexneri M90T is established in an unambiguous multidisciplinary approach. Chemical and spectroscopical investigation of the de-acetylated LPS showed that the inner core structure is characterized by a L,D-Hep-(1 -->7)-L,D-Hep-(1 -->3)-L,D-Hep-(1 -->5)-[Kdo-(2 -->4)]-Kdo sequence that is the common structural theme identified in Enterobacteriaceae. In particular, in S. flexneri M90T serotype 5 LPS, a glucosamine residue is additionally sitting at O-7 of the last heptose whereas the outer core is characterized by glucose and galactose residues. Also, in order to exactly define the position of glycine that is an integral constituent of the core region of the LPS, we created a S. flexneri M90T delta galU mutant and studied its LOS. In this way it was possible to establish that glycine is sitting at O-6 of the second heptose in the inner core.     

Factors controlling acid tolerance of Listeria monocytogenes: effects of nisin and other ionophores.

The acid tolerance of a Listeria monocytogenes serotype 4b strain was studied by measuring its ability to survive at an acidic pH at 37 degrees C. The acid tolerance of L. monocytogenes was much lower than those of Escherichia coli O157:H7 and Shigella flexneri strains. This observation suggested a higher infective dose for L. monocytogenes than E. coli O157:H7 and Shigella. The susceptibility of L. monocytogenes to acidic pH was dependent upon growth medium pH and growth phase of the culture. Nisin and some other ionophores reduced the acid tolerance of both stationary-phase and log-phase cultures of L. monocytogenes. These studies indicated that nisin might be a useful candidate for controlling acid tolerance of L. monocytogenes.     

环介导等温扩增联合横向流动试纸条可视化检测志贺氏菌

【目的】将环介导等温扩增技术(LAMP)与横向流动试纸条(LFD)联合应用,建立一种可应用于志贺氏菌快速检测的LAMP-LFD技术。【方法】以福氏志贺氏菌的侵袭性质粒抗原H(ipa H)基因为检测靶标设计3对特异性引物(其中上游内引物Sfl-ipa H-FIP由生物素标记),进行LAMP反应;同时设计1条异硫氰酸荧光素(FITC)标记的探针Sfl-ipa H-HP,与获得的LAMP产物进行特异性杂交,杂交产物经LFD完成检测。【结果】优化后的LAMP反应条件为63°C 40 min,加上LFD结果判读共需50 min。LAMP-LFD方法能够特异性检测出福氏志贺氏菌,而对肠炎沙门氏菌等其它4种导致腹泻的致病菌和创伤弧菌等5种常见食物源性致病菌,以及4株不同大肠杆菌的检测结果呈阴性。该方法针对福氏志贺氏菌的检测灵敏度为1.0×10~2 CFU/m L或4 CFU/反应,针对人工污染鲤鱼肠组织的检测灵敏度是5.0×10~2 CFU/m L,是以LAMP外引物Sfl-ipa H-F3/Sfl-ipa H-B3的常规PCR方法的100倍。【结论】建立的LAMP-LFD技术具有操作简单、检测快速准确、检测成本低等优点,有望在志贺氏菌的常规监测和即时检测中被普及使用。     

112株志贺菌菌群分布和药敏特点分析

目的研究本地区2001年至2005年志贺菌菌群分布及其药敏特点,以指导临床合理抗菌治疗。方法经大便培养筛选志贺菌,用生化和血清学方法鉴定菌群和血清型,采用K-B法检测病原菌耐药性。结果在112例细菌性痢疾患者中,男女比例相似,年龄分布以婴幼儿最高,临床表现不典型者较多,菌群分布以福氏志贺菌最多,F2b为优势血清型,对抗菌药物敏感性差异有显著性。结论近5年来本地区细菌性痢疾患者发病特点有年龄差异,菌群仍以福氏志贺菌为主,血清型以F2b为主,第3代头孢菌素是治疗细菌性痢疾最佳的抗菌药物。