Shigella

New Microbiol, 2004 Oct, 27(4), 381 - 9
Occurrence, characterisation and detection of potential virulence determinants of emerging aquatic bacterial pathogens from the Philippines and Thailand; Maluping RP et al.; Strains of Aeromonas spp., 'non-cholera vibrios' (NCVs) and Plesiomonas shigelloides isolated from aquatic environments, fish and human diarrhoeal cases in the Philippines and Thailand were characterised for potential virulence markers . Thus, the production of cytotoxin, cell-associated and cell-free haemolysin and their capacity to adhere to human intestinal (Henle 407) cells in vitro was investigated . In addition, the occurrence of tlh and tdh haemolysin genes and urease activity among V . parahaemolyticus strains was investigated . The results showed that strains recovered from clinical sources (human and fish) produced these virulence factors, whereas these are absent in environmental strains.

Nucleic Acids Res . 2005 Jan 07;33(1):e3.
ArrayOme: a program for estimating the sizes of microarray-visualized bacterial genomes; Ou HY et al.; ArrayOme is a new program that calculates the size of genomes represented by microarray-based probes and facilitates recognition of key bacterial strains carrying large numbers of novel genes . Protein-coding sequences (CDS) that are contiguous on annotated reference templates and classified as 'Present' in the test strain by hybridization to microarrays are merged into ICs (ICs) . These ICs are then extended to account for flanking intergenic sequences . Finally, the lengths of all extended ICs are summated to yield the 'microarray-visualized genome (MVG)' size . We tested and validated ArrayOme using both experimental and in silico-generated genomic hybridization data . MVG sizing of five sequenced Escherichia coli and Shigella strains resulted in an accuracy of 97-99%, as compared to true genome sizes, when the comprehensive ShE.coli meta-array gene sequences (6239 CDS) were used for in silico hybridization analysis . However, the E.coli CFT073 genome size was underestimated by 14% as this meta-array lacked probes for many CFT073 CDS . ArrayOme permits rapid recognition of discordances between PFGE-measured genome and MVG sizes, thereby enabling high-throughput identification of strains rich in novel genes . Gene discovery studies focused on these strains will greatly facilitate characterization of the global gene pool accessible to individual bacterial species.

Appl Environ Microbiol, 2005 Jan, 71(1), 320 - 5
Inactivation of Escherichia coli O157:H7 in Simulated Human Gastric Fluid; Tamplin ML; Human disease caused by Escherichia coli O157:H7 is a function of the number of cells that are present at potential sites of infection and host susceptibility . Such infectious doses are a result, in part, of the quantity of cells that are ingested and that survive human host defenses, such as the low-pH environment of the stomach . To more fully understand the kinetics of E . coli O157:H7 survival in gastric fluid, individual E . coli O157:H7 strains were suspended in various media (i.e., saline, cooked ground beef {CGB}, and CGB containing a commercial antacid product {CGB+A}), mixed at various proportions with simulated human gastric fluid (SGF), and then incubated at 37 degrees C for up to 4 h . The highest inactivation rate among nine E . coli O157:H7 strains was observed in saline . Specifically, the average survival rates in 100:1 and 10:1 proportions of SGF-saline were -1.344 +/- 0.564 and -0.997 +/- 0.388 log(10) CFU/h, respectively . In contrast, the average inactivation rate for 10 E . coli O157:H7 strains suspended in 10:1 SGF-CGB was -0.081 +/- 0.068, a rate that was 12-fold lower than that observed for SGF-saline . In comparison, the average inactivation rate for Shigella flexneri strain 5348 in 100:1 and 10:1 SGF-saline was -8.784 and -17.310, respectively . These latter inactivation rates were 7- to 17-fold higher than those for E . coli O157:H7 strains in SGF-saline and were 4-fold higher than those for E . coli O157:H7 strains in SGF-CGB . The survival rate of E . coli O157:H7 strain GFP80EC increased as the dose of antacid increased from one-half to twice the prescribed dose . A similar trend was observed for the matrix pH over the range of pH 1.6 to 5.7, indicating that pH is a primary factor affecting E . coli O157:H7 survival in SGF-CGB+A . These results can be used in risk assessment to define dose-response relationships for E . coli O157:H7 and to evaluate potential surrogate organisms.

Yi Chuan, 2004 Jul, 26(4), 495 - 8
{Construction of SW480 Cell Model Identifying Shigella Virulent Genes.}; Yao X et al.; Signature-tagged mutagenesis (STM) is a novel technology with high throughput screening ability to identify virulent genes of pathogen in vivo.An appropriate animal or cell line model is one of prerequisites by exploiting this technique.In order to apply STM to Shigella flexneri, RC426 was constructed as an attenuated mutant with chloramphenicol resistance and aroA and virG genes inactivated by homologous recombination; Another attenuated strain T32 was used as an oral S.flexneri 2a vaccine due to a spontaneous deletion in three loci (ipaBCDA, invA and virG) on the virulence plasmid.The wild type strain 2457T had the invasion ability into host cells.The three strains, RC426, T32 and 2457T, were mixed together to invade colon cancer cell line SW480, and the distinct strains were recovered and counted from cell lysates of invaded SW480 in different time.The results showed that there were statistically significant differences between the amounts of two attenuated strains recovered and that of virulent strain within 12h invasion, indicating SW480 was a suitable cell model for applying STM to screen virulent genes of Shigella flexneri.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Nov-Dec, (6), 13 - 8
{Choice of informative characteristics for the characterization of the adaptive process in the population of rod-shaped bacteria}; Antimicrobial resistance of Shigella flexneri serotypes in Israel during a period of three years: 2000-2002; Central Laboratories of the Ministry of Health, Jerusalem, Israel . valentin.vasilev@eliav.health.gov.il

This is a surveillance study of the antimicrobial resistance of the S . flexneri group in the context of its serotype diversity . It includes 1422 isolates, which were sent to the National Shigella Reference Centre (NSRC) by hospitals and outpatient clinics in Israel during a 3-year period (2000-2002) . The strains were identified and classified according to the prevalence and antigenic structure of their serotypes . All samples were checked for resistance to ampicillin (AMP), trimethoprim-sulphamethoxazole (TMP-SMX), ceftriaxone (CRO), tetracycline (TE), nalidixic acid (NAL), and chloramphenicol (C) by the disk diffusion method of Bauer et al . There were significant differences in their resistance to the individual antimicrobials with resistance to AMP, TE and C being lower among the strains of serotype 6 than among those of serotypes 2a and 1b . The resistant phenotypes were also serotype-specific . The similarities both in individual and in phenotype resistance between the rare and the prevalent serotypes (but not serotype 6) may be attributed to their antigenic relatedness . The serospecificity of the antimicrobial resistance was not affected by external factors such as seasonality and source (hospital or outpatient laboratory) of the isolates, and the age and sex of the patients . The serotype-specific approach can assist in properly assessing the problem of the antimicrobial resistance of the Shigella flexneri group and may prove useful for the empirical therapy of shigellosis . The observed interdependency between resistance and the antigenic specificity and relatedness of the S . flexneri serotypes requires additional investigation.

Proteins . 2004 Dec 28; {Epub ahead of print}
Crystal structure of the copper homeostasis protein (CutCm) from Shigella flexneri at 1.7 A resolution: The first structure of a new sequence family of TIM barrels; Zhu DY et al.; No abstract.

Sci China C Life Sci, 2004 Dec, 47(6), 494 - 502
Screening and identification of Shigella flexneri 2a virulence-related genes induced after invasion of epithelial cells; Shi Z et al.; An in vivo expression technology (IVET) was applied to screen S . flexneri 2a genes induced after invasion of epithelial cells, and virulence-related genes were further identified by mutational analysis . Thirteen intracellular induced genes were identified with a HeLa cell infection model . Of these, two were identified as alkylation-related genes; one was related to metabolism; one encoded a transcriptional regulator; three were identified as insertion elements; three appeared to be antisense to genes involved in the transmethylation, biosynthesis, and phosphotransferase system; and three were predicted to encode polypeptides with unknown functions . Intracellular survival assays showed that the mutants of alkA, citC and wcaJ genes had lower capability of intracellular replication or survival than the wild-type strain . The results indicated that alkA, citC and wcaJ genes could take part in the intracellular survival or replication of S . flexneri 2a and the capability of intracellular survival or replication could be one of the major virulence elements . However, the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay . Very possibly, yaiC gene was involved in the other mechanism of S . flexneri virulence . This study might lead to a better understanding of the intracellular survival or proliferation process of S . flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen.

Infect Immun, 2005 Jan, 73(1), 504 - 13
Virulent Shigella flexneri causes damage to mitochondria and triggers necrosis in infected human monocyte-derived macrophages; Koterski JF et al.; Shigella flexneri is a gram-negative bacterium that causes bacillary dysentery in humans that is characterized by an acute inflammatory response of the colon . The fate of phagocytes that are infected in vitro with virulent Shigella has been the subject of some investigation and debate . In this study we found that virulent Shigella caused a rapid increase in the cell membrane permeability of infected human monocyte-derived macrophages (HMDM) but not in the cell membrane permeability of monocytes, as demonstrated by the uptake of fluorescent vital dyes . Within 2 h of infection, 59% +/- 6% of the HMDM and </=4% of the monocytes were stained with propidium iodide . Treatment of the cells with the inhibitors of caspases YVAD and zVAD, the antioxidants N-acetyl-l-cysteine and butylated hydroxyanisole, or an inhibitor of NADPH oxidase, diphenyleniodonium, did not alter the infection outcome . Importantly, we found that virulent Shigella caused a rapid drop in the ATP level to about 50% in infected HMDM . Furthermore, using a combination of fluorescent vital dyes and mitochondrial membrane potential-sensitive dyes, we observed that cells that exhibited a permeable cell membrane were not stained by the mitochondrion-specific dyes, indicating that the mitochondrial membrane potential was lost in these cells . We also observed infected cells that were not stained with either type of dye, indicating that the loss of the mitochondrial membrane potential preceded the increase in cell membrane permeability . Taken together, our studies showed that virulent Shigella flexneri targets the host cell mitochondria for destruction . This activity may account for the necrotic cell death precipitated by these pathogens.

Infect Immun, 2005 Jan, 73(1), 258 - 67
Construction and characterization of bivalent Shigella flexneri 2a vaccine strains SC608(pCFAI) and SC608(pCFAI/LTB) that express antigens from enterotoxigenic Escherichia coli; Ranallo RT et al.; An invasive strain of Shigella flexneri 2a (SC608) has been developed as a vector for the expression and delivery of heterologous antigens . SC608 is an aspartate semialdehyde dehydrogenase (asd) derivative of SC602 (icsA iuc), a well-characterized live attenuated vaccine strain which has undergone several clinical trials in human volunteers . When administered orally at a single 10(4) (CFU) dose, SC602 is both immunogenic and efficacious against shigellosis . Using asd-based plasmid vectors, we designed SC608 to express the enterotoxigenic Escherichia coli (ETEC) fimbrial subunit CfaB (CFA/I structural subunit) alone or in combination with the E . coli B subunit of heat-labile enterotoxin (LTB) . The expression of each heterologous protein in SC608 was verified by immunoblot analysis . Each strain was comparable to the parent strain, SC602, in a HeLa cell invasion assay . After intranasal immunizations of guinea pigs, serum and mucosal immune responses were detected against both Shigella lipopolysaccharide and heterologous ETEC antigens by enzyme-linked immunosorbent assay and ELISPOT analysis . All immunized animals were subsequently protected against a challenge with wild-type S . flexneri 2a in a keratoconjunctivitis Sereny test . Serum antibodies generated against LTB and CfaB demonstrated antitoxin and agglutination activities, respectively . These results suggest that CfaB and LTB expressed in SC608 retain important conformational epitopes that are required for the generation of antibodies that have functional activities . These initial experiments demonstrate that a fully invasive Shigella vaccine strain can be engineered to deliver antigens from other diarrheal pathogens.

Cell Microbiol, 2005 Jan, 7(1), 115 - 127
Intravenous infection of virulent shigellae causes fulminant hepatitis in mice; Martino MC et al.; Summary Shigella spp . are pathogenic bacteria responsible for bacillary dysentery in humans . The major lesions in colonic mucosa are intense inflammation with apoptosis of macrophages and release of pro-inflammatory cytokines . The study of shigellosis is hindered by the natural resistance of rodents to oral infection with Shigella . Therefore, animal models exploit other routes of infection . Here, we describe a novel murine model in which animals receive shigellae via the caudal vein . Mice infected with 5 x 10(6) (LD(50)) virulent shigellae died at 48 h post infection, whereas animals receiving non-invasive mutants survived . The liver is the main target of infection, where shigellae induce microgranuloma formation . In mice infected with invasive bacteria, high frequency of apoptotic cells is observed within hepatic microgranulomas along with significant levels of mRNA for pro-inflammatory cytokines such as IL-1beta, IL-18, IL-12 and IFN-gamma . Moreover, in the blood of these animals high levels of IL-6 and transaminases are detected . Our results demonstrate the intravenous model is suitable for pathogenicity studies and useful to explore the immune response after Shigella infection.

Microbiol Immunol, 2004, 48(12), 965 - 9
Release of Shiga Toxin by Membrane Vesicles in Shigella dysenteriae Serotype 1 Strains and In Vitro Effects of Antimicrobials on Toxin Production and Release; Dutta S et al.; Effects of various antimicrobials on in vitro Shiga toxin production and release by Shigella dysenteriae serotype 1 was investigated in this study with particular reference to the role of outer membrane vesicles in toxin release by the organism . Five antimicrobials, namely nalidixic acid, ciprofloxacin, norfloxacin, fosfomycin and mitomycin C, were chosen for the study and the toxin titre was measured by the reverse passive latex agglutination (RPLA) method using an available kit . Only mitomycin C was found to induce production of Shiga toxin in the bacteria and its release by outer membrane vesicles . The highest titre of toxin was obtained in vesicle fraction suggesting that the vesicles play an important role in the release of Shiga toxin from periplasmic space by the organism.

Arch Med Res, 2004 Sep-Oct, 35(5), 427 - 34
Role of 57 kDa major antigenic component of Shigella dysenteriae outer membrane proteins in induction of major histocompatibility complex II-restricted T-cell response; Bagchi AK et al.; BACKGROUND: In the past, many Shigella surface antigens were used to activate both T and B lymphocytes but failed to induce antigen-specific responses in Shigellosis . Our objective was to identify in vitro T-cell components using 57 kDa major antigenic fraction of Shigella dysenteriae 1 (IPC-31) outer membrane proteins (OMPs) in modulating specific T-cell subset responses against Shigellosis . METHODS: Antigen-specific T- and B-cell activation was studied in immunized Balb/c mice against 57 kDa antigen by proliferative responses using {(3)H}-thymidine incorporation and avidin-biotin complex (ABC) peroxidase staining for CD4, CD8, CD3, CD22, and CD25 followed by IL-2 and IL-4 estimation . Macrophage functional assays for migration inhibition factors (MIF) and superoxide (O(2)(-)) anions were also performed against 57 kDa antigen, whole OMPs, and phytohemagglutinin (PHA) stimulation . RESULTS: Greater increase of lymphocyte proliferation was observed after 57 kDa antigen stimulation than post-OMP and -PHA stimulation . Proportionately, CD4(+) and CD25(+) expression of total CD3(+) T-cells was significantly dominant (p >0.05) over CD8(+) T-cells . On day 7 of this stimulation, it was found to increase % MIF and O(2)(-) anions with decrease of IL-2 leading to activation of MHC-II antigens . Later, on day 28 of immunization, IL-2 levels were more increased than on days 7 and 14 but insignificant with non-immunized mice stimulated with 57 kDa . Levels of IL-2 were also noted with low degree of internalization to its IL-2R receptors rather than to IL-4 receptors . In parallel, expression of CD22 was also recorded higher in this stimulation than in PHA, indicating a T-cell-dependent humoral response . CONCLUSIONS: Our results suggested that 57 kDa major antigenic OMP is immunogenic for MHC II-restricted T-cell response to acquire host defense against Shigella infection.

J Health Popul Nutr, 2004 Sep, 22(3), 322 - 30
The importance of engaging policy-makers at the outset to guide research on and introduction of vaccines: the use of policy-maker surveys; DeRoeck D; Face-to-face surveys of policy-makers and other influential leaders are a useful tool to identify, at an early stage, (a) major issues regarding the introduction of a new vaccine, (b) persons and groups in a country who play a major decision-making or influential role in the introduction of vaccines, (c) potential obstacles to the introduction of vaccines, and (d) data-needs of policy-makers to overcome these obstacles . By surveying the opinions and beliefs of those who will make or influence decisions on whether to introduce a new vaccine, these studies can help ensure that research activities respond to the needs of policy-makers in countries endemic for the target diseases . These surveys can also inform vaccine-introduction strategies by identifying financially and politically feasible means of distributing, targeting, and financing the vaccines . This paper describes the methodology used in conducting such surveys and discusses methodological issues . It also presents lessons learnt from two policy-maker surveys carried out in several Asian countries in regard to new-generation vaccines against cholera, typhoid fever, and shigellosis; and future vaccines against dengue fever/dengue haemorrhagic fever.

J Health Popul Nutr, 2004 Sep, 22(3), 293 - 303
Sociobehavioural research methods for the introduction of vaccines in the Diseases of the Most Impoverished Programme; Kaljee LM et al.; Participation in vaccination campaigns worldwide, particularly the Expanded Programme on Immunization, has increased significantly in recent years . However, there remain multiple and integrated behavioural, sociocultural and political-economic barriers to vaccination . The Diseases of the Most Impoverished (DOMI) Programme has undertaken shigellosis disease-burden studies and oral cholera and typhoid Vi polysaccharide vaccine trials in seven Asian countries . As part of these projects, sociobehavioural studies have been undertaken to determine the potential demand for vaccines for these diseases and the obstacles and enabling factors that may affect acceptance, delivery, and use of vaccines . A theoretical model of acceptance of vaccination and a triangulation of qualitative and quantitative methods have been used for fully elucidating the range of issues relating to vaccination for shigellosis, cholera, and typhoid fever . In this paper, the theoretical and methodological basis of the DOMI projects has been reviewed in a context of current sociobehavioural research on the acceptability and desirability of vaccination.

J Health Popul Nutr, 2004 Sep, 22(3), 232 - 9
Multidisciplinary studies of disease burden in the Diseases of the Most Impoverished Programme; Deen JL et al.; With limited healthcare resources, rational prioritization of healthcare interventions requires knowledge and analysis of disease burden . In the absence of actual disease-burden data from less-developed countries, various types of morbidity and mortality estimates have been made . Besides having questionable reliability, these estimates do not capture the full burden of a disease since they provide only the number of cases and deaths . The modelling methods that include disability are more comprehensive but are difficult to understand, and their reliability is affected by baseline approximations . To provide policy-makers with information needed for rational decision-making, the Diseases of the Most Impoverished (DOMI) Programme of the International Vaccine Institute has used a multidisciplinary approach to describe the burden of disease due to typhoid fever, shigellosis, and cholera . Recognizing the relative advantages and disadvantages of various methodologies, the programme employs passive clinic-based surveillance in defined communities to provide prospective data . The prospective data are complemented with retrospectively-collected information from existing sources, frequently less accurate and complete but readily available for the whole population over extended periods . To create a more complete picture, economic and qualitative studies specific to each disease are incorporated in these prospective studies . The goal is to achieve a more complete and realistic picture by combining the results of these various methodologies, acknowledging the strengths and limitations of each . These projects also build in-country capacity in terms of treatment, diagnosis, epidemiology, and data management.

J Health Popul Nutr, 2004 Sep, 22(3), 223 - 31
Translational research to assist policy decisions about introducing new vaccines in developing countries; Clemens JD et al.; Few new-generation vaccines have found their way into public-health programmes for the poor in developing countries, and for those that have, delays of years or even decades after their licensure and introduction in industrialized countries have been the rule . Financial constraints and political obstacles have played major roles in delaying the introduction of the vaccines . Also contributing to this situation has been a dearth of needed research . While past analyses have identified inadequate support for conducting Phase 1 studies as an obstacle, other types of translational research are also needed . Vaccines may perform less well in impoverished populations in the developing world than in more affluent populations . Consequently, Phase 2 and Phase 3 trials of new vaccines in developing countries are a second essential type of translational research needed for the introduction of vaccines in developing countries . Moreover, even for vaccines that have performed well in pre-licensure human trials in developing countries, doubts often remain about whether the local disease burden justifies introduction of vaccine, whether the vaccine will be cost-effective, and whether introduction of vaccine will be programmatically feasible, acceptable, and financially sustainable . Because these residual doubts constitute obstacles to the introduction of vaccine, a third type of translational research is needed to provide this evidence required for policy . In this paper, these three types of translational research are illustrated with projects being undertaken in the Diseases of the Most Impoverished Programme . The Programme is conducting translational research to accelerate the rational introduction of new vaccines against cholera, shigellosis, and typhoid fever in developing countries affected by these diseases.

Immunology, 2005 Jan, 114(1), 94 - 100
Porin of Shigella dysenteriae enhances Toll-like receptors 2 and 6 of mouse peritoneal B-2 cells and induces the expression of immunoglobulin M, immunoglobulin G2a and immunoglobulin A; Ray A et al.; Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptors TLR2 and TLR6, by 1.8-fold and twofold, respectively, in peritoneal cavity B-2 cells from C57BL/6 mice, implicating that the co-expression of TLR2 and TLR6 occurs as a combinatorial repertoire in response to porin . Among the two key TLRs, TLR2 and TLR4, which are primarily responsible for recognizing the majority of bacterial products, TLR2 alone participates in porin recognition . TLR2 expression was increased on B-2 cells, whereas the expression of TLR4 remained unaffected . Besides TLRs, mRNA for myeloid differentiation factor 88 (MyD88), an effector molecule associated with the TLR-mediated response, was enhanced by twofold, suggesting its involvement in the activity of porin . The B-2 cells showed a 1.8-fold increase in mRNA expression of the signalling molecule, nuclear factor-kappa B (NF-kappaB), in the presence of porin . Porin treatment of B-2 cells selectively up-regulated the expression of the costimulatory molecule, CD86, by 4.4-fold . Porin induced the cell-surface expression of immunoglobulin (Ig)M, of IgG2a preferentially among the IgG subclasses, and of IgA, on B-2 cells . The porin-mediated inductions of IgG2a and IgA were augmented by interleukin-6 on B-2 cells, by 2.7- and 1.6-fold, respectively.

J Bacteriol, 2005 Jan, 187(1), 107 - 13
A sensor of the two-component system CpxA affects expression of the type III secretion system through posttranscriptional processing of InvE; Mitobe J et al.; The chief function of the Cpx two-component system is perceiving various cell envelope stresses, but CpxR is also known to regulate the expression of the type III secretion system (TTSS) of Shigella sonnei through transcription of the primary regulator virF . Here, we have isolated novel cpxA mutants that exhibited decreased TTSS expression from Escherichia coli HW1273, which carries the virulence plasmid of S . sonnei . The cpxA deletion strain of HW1273 expressed beta-galactosidase activity levels from the virF-lacZ fusion similar to those of HW1273 . However, the second regulator InvE (VirB) and the TTSS component IpaB proteins were apparently expressed at a low level . In the cpxA strain, beta-galactosidase activity levels from the invE-lacZ transcriptional fusion remained similar to those of HW1273, whereas the beta-galactosidase activity level from the translational fusion of invE-lacZ was reduced to 21% of that of HW1273 . Therefore, the deletion of the cpxA gene influenced TTSS expression chiefly at the posttranscriptional processing of InvE . In addition, the cpxA deletion strain of S . sonnei showed the same phenotype . These results indicate that the Cpx two-component system is involved in virulence expression through posttranscriptional processing of the regulatory protein InvE, a novel feature of the Cpx two-component system in posttranscriptional processing and virulence expression of Shigella.

Indian J Med Res, 2004 Nov, 120(5), 454 - 62
Shigellosis : challenges & management issues; Sur D et al.; Infectious diseases kill about 11 million children each year while acute diarrhoeal diseases account for 3.1 million deaths in children under 5 yr of age, of which 6,00,000 deaths annually are contributed by shigellosis alone . Shigellosis, also known as acute bacillary dysentery, is characterized by the passage of loose stools mixed with blood and mucus and accompanied by fever, abdominal cramps and tenesmus . It may be associated with a number of complications of which haemolytic uraemic syndrome is the most serious . Shigellosis is caused by Shigella spp . which can be subdivided into four serogroups namely S.sonnei, S.boydii, S.flexneri and S.dysenteriae . Organisms as low as 10-100 in number can cause the disease . Shigellosis can occur in sporadic, epidemic and pandemic forms . Epidemics have been reported from Central American countries, Bangladesh, Sri Lanka, Maldives, Nepal, Bhutan, Myanmar and from the Indian subcontinent, Vellore, eastern India and Andaman and Nicobar islands . Plasmid profile of shigellae in Kolkata has shown a correlation between presence of smaller plasmids and shigellae serotypes- indicating epidemiological changes of the species . Diagnosis of shigellosis is essentially clinical . Laboratory diagnosis includes stool culture and polymerase chain reaction (PCR) . Treatment includes use of an effective antibiotic, rehydration therapy (if there is dehydration) and appropriate feeding during and after an episode of shigellosis . Hand-washing is the single most important strategy for prevention of transmission of shigellosis from person to person . A safe and effective vaccine should be developed against the more important circulating strains i.e., S . dysenteriae type 1 and S . flexneri 2a.

J Huazhong Univ Sci Technolog Med Sci, 2004, 24(4), 400 - 3
Experimental study on the antibacterial effect of origanum volatile oil on dysentery bacilli in vivo and in vitro; Liao F et al.; To observe the germistatic and germicidal effects of origanum volatile oil (OVI) on the dysentery bacteria, the abdominal cavity of mice was infected with Shigella sonne (Sh . sonnei) and Shigella flexneri (Sh . flexneri) F2a . After OVI was given to the mice via gastric lavage, the effects of OVI on the infected mice were observed . The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) for dysentery bacteria were determined in vitro . The results showed that origanum volatile oil showed obvious protective effect on mice infected with Sh . sonnei and Sh . flexneri F2a and it had germistatic and germicidal effects on dysentry bacteria . We are led to conclude that origanum volatile oil is an effective medicine against the infection of dysentery bacteria.

J Clin Microbiol, 2004 Dec, 42(12), 5849 - 53
Evaluation of multiplex PCRs for diagnosis of infection with diarrheagenic Escherichia coli and Shigella spp; Aranda KR et al.; We have developed two multiplex PCR assays that detect typical and atypical enteropathogenic Escherichia coli (EPEC) isolates, enteroaggregative E . coli (EAEC) isolates, enterotoxigenic E . coli (ETEC) isolates, enteroinvasive E . coli (EIEC) isolates, Shiga toxin-producing E . coli (STEC) isolates, and Shigella spp . The targets selected for each group were eae and bfpA for EPEC isolates, the target of probe CVD432 for EAEC isolates, the genes encoding heat-labile and heat-stable toxins for ETEC isolates, stx(1) and stx(2) for STEC isolates, and ipaH for EIEC isolates and Shigella spp . These PCRs were specific and sensitive for rapid detection of target isolates in stools . Among 150 stool specimens from the acute diarrhea tested, 9 samples (6%) had atypical EPEC, 9 (6%) had typical EPEC, 7 (4.7%) had EAEC, 3 (2%) had EIEC, 3 (2%) had Shigella spp., and 1 (0.7%) had an O26 STEC strain; we also detected mixed infections, 2 (1.3%) with EAEC and Shigella spp., 1 (0.7%) with atypical and typical EPEC strains, and another with atypical EPEC and EAEC strains . One of the multiplex PCRs directly applied to 36 stool specimens correctly identified 100% of EPEC and EAEC isolates.

J Clin Microbiol, 2004 Dec, 42(12), 5745 - 50
Diarrhea caused by rotavirus in children less than 5 years of age in Hanoi, Vietnam; Nguyen TV et al.; Group A rotaviruses are the major cause of diarrhea in young children worldwide . From March 2001 to April 2002, 836 children less than 5 years of age were investigated in Hanoi, Vietnam . This included 587 children with diarrhea and 249 age-matched controls . Group A rotavirus was identified in 46.7% of the children with diarrhea and 3.6% of the controls, which was a significant difference . Within the diarrhea group, the highest prevalence was seen in children from 13 to 24 months of age, and the prevalence was higher in males than in females . The symptoms of acute diarrhea caused by rotavirus were watery diarrhea, vomiting, fever, and dehydration . A higher prevalence of rotavirus detection was obtained for children who had all of these symptoms, followed by those who had diarrhea with vomiting-dehydration, fever-dehydration, and dehydration . The high rates occurred from September to December, although the infection was encountered all year round . In 58 patients (21.2% of the rotavirus-infected children), rotavirus infection was detected in association with either diarrheagenic Escherichia coli or Shigella spp . The most frequent combinations were rotavirus-enteroaggregative E . coli and rotavirus-enteropathogenic E . coli . At least one enteropathogen was identified from about 64% percent of the samples . The bacterial infection may not have given rise to clinical symptoms of such severity . The present study demonstrates the burden of rotavirus diarrhea in Hanoi, Vietnam . Continuous surveillance of diarrhea caused by rotavirus in young children would play an important role in diagnosis, treatment, and prophylaxis in order to improve the health of children in Vietnam.

Science . 2004 Dec 2; {Epub ahead of print}
Escape of Intracellular Shigella from Autophagy; Ogawa M et al.; The degradation of undesirable cellular components or organelles, including invading microbes, by autophagy is crucial for cell survival . Here, Shigella, an invasive bacteria, was found to be able to escape autophagy by secreting IcsB via the type III secretion system . Mutant bacteria lacking IcsB were trapped by autophagy during multiplication within the host cells . IcsB did not directly inhibit autophagy . Rather, Shigella VirG, a protein required for intracellular actin-based motility, induced autophagy by binding to the autophagy protein, Atg5 . In non-mutant Shigella, this binding is competitively inhibited by IcsB binding to VirG.

Expert Opin Biol Ther, 2004 Dec, 4(12), 1939 - 51
Vaccines against cholera, typhoid fever and shigellosis for developing countries; Acosta CJ et al.; Enteric diseases, such as cholera, typhoid fever and shigellosis, still produce a significant burden, especially among the poor in countries where these illnesses are endemic . Older-generation, parenteral, whole-cell vaccines against cholera and typhoid fever were abandoned in many countries as public health tools because of problems with insufficient protection and/or inadequate safety profiles . Modern-generation licensed vaccines are available for cholera and typhoid fever, but are not widely used by those in greatest need . A number of experimental candidates exist for all three diseases . Future research should focus on generating the evidence necessary to obtain a consensus on the deployment of existing vaccines against cholera and typhoid fever, and on clinical evaluation of pipeline vaccine candidates against all three diseases.

Protein Eng Des Sel, 2004 Oct, 17(10), 721 - 9 Epub 2004 Nov 19.
Bacterial lipid modification of proteins for novel protein engineering applications; Kamalakkannan S et al.; Functioning of proteins efficiently at the solid-liquid interface is critical to not only biological but also modern man-made systems such as ELISA, liposomes and biosensors . Anchoring hydrophilic proteins poses a major challenge in this regard . Lipid modification, N-acyl-S-diacylglyceryl-Cys, providing an N-terminal hydrophobic membrane anchor is a viable solution that bacteria have successfully evolved but remains unexploited . Based on the current understanding of this ubiquitous and unique bacterial lipid modification it is possible to use Escherichia coli, the popular recombinant protein expression host, for converting a non-lipoprotein to a lipoprotein with a hydrophobic anchor at the N-terminal end . We report two strategies applicable to non-lipoproteins (with or without signal sequences) employing minimal sequence change . Taking periplasmic Shigella apyrase as an example, its signal sequence was engineered to include a lipobox, an essential determinant for lipid modification, or its mature sequence was fused to the signal sequence of abundant outer membrane lipoprotein, Lpp . Lipid modification was proved by membrane localization, electrophoretic mobility shift and mass spectrometric analysis . Substrate specificity and specific activity measurements indicated functional integrity after modification . In conclusion, a convenient protein engineering strategy for converting non-lipoprotein to lipoprotein for commercial application has been devised and tested successfully.

Ann Rheum Dis . 2004 Nov 18; {Epub ahead of print}
Reactive arthritis attributable to Shigella infection: a clinical and epidemiological nation-wide study; Hannu T et al.; OBJECTIVES: To study the incidence and clinical picture of Shigella-associated reactive arthritis (ReA) and the arthritogenicity of various Shigella species in the population . METHODS: A questionnaire on enteric and extraintestinal, especially musculoskeletal, symptoms went to 278 consecutive patients with Shigella-positive stool culture and to 597 controls . Analysis of self- reported musculoskeletal symptoms was supplemented with clinical examination of those subjects with recent symptoms . RESULTS: Of the patients, 14 (7%) had ReA, and a further 4 (2%) other reactive musculoskeletal symptoms (tendinitis, enthesopathy, or bursitis) . Of the 14 ReA patients, all adults, 10 had S . sonnei, 3 S . flexneri, and one S . dysenteriae infection . HLA-B27 was positive in 36% of the ReA patients . One control subject had ReA . In the patients with Shigella infection, the odds ratio for developing ReA was 16.2 (95% CI 2.1-123.9), P=0.001 . CONCLUSIONS: ReA occurred in 7% of patients following Shigella infection, with an annual incidence of 1.3/1 000 000 in Finland . Besides S . flexneri, also S . sonnei and S . dysenteriae are able to trigger ReA.

J Clin Microbiol, 2004 Nov, 42(11), 5264 - 9
Occurrence of extended-spectrum beta-lactamases in members of the genus Shigella in the Republic of Korea; Kim S et al.; A nationwide survey was carried out in Korea to assess the prevalence of Shigella strains producing extended-spectrum beta-lactamases (ESBLs) . From 1991 to 2002, 5,911 clinical strains were isolated and screened for resistance to extended-spectrum cephalosporins . Twenty of the Shigella isolates were ESBL positive, based on the synergistic effects between clavulanate and selected beta-lactams (ceftazidime and cefotaxime) . Nucleotide sequence analysis of these isolates revealed that they harbored bla(TEM-19) (eight isolates), bla(TEM-15) (five isolates), bla(TEM-52) (six isolates), bla(TEM-17) (one isolate), bla(TEM-20) (one isolate), and bla(CTX-M-14) (three isolates) . All the ESBL-encoding genes in this study were carried in conjugable plasmids . Thus, TEM-19, TEM-15, TEM-52, and CTX-M-14 beta-lactamases can be considered common Korean ESBL types in Shigella sonnei and are probably transmitted through interspecies spread between medical facilities and the community in Korea . This is the first report of the presence of TEM-17, TEM-19, and TEM-20 in Korea and in S . sonnei.

Microbiology, 2004 Nov, 150(Pt 11), 3631 - 45
Identification of Escherichia coli O157 : H7 genes influencing colonization of the bovine gastrointestinal tract using signature-tagged mutagenesis; Dziva F et al.; Enterohaemorrhagic Escherichia coli (EHEC) cause acute gastroenteritis in humans that may be complicated by life-threatening systemic sequelae . The predominant EHEC serotype affecting humans in the UK and North America is O157 : H7 and infections are frequently associated with contact with ruminant faeces . Strategies to reduce the carriage of EHEC in ruminants are expected to lower the incidence of human EHEC infections; however, the molecular mechanisms underlying persistence of EHEC in ruminants are poorly understood . This paper reports the first comprehensive survey for EHEC factors mediating colonization of the bovine intestines by using signature-tagged transposon mutagenesis . Seventy-nine E . coli O157 : H7 mutants impaired in their ability to colonize calves were isolated and 59 different genes required for intestinal colonization were identified by cloning and sequencing of the transposon insertion sites . Thirteen transposon insertions were clustered in the locus of enterocyte effacement (LEE), which encodes a type III protein secretion system required for the formation of attaching and effacing lesions on intestinal epithelia . A putative structural component of the apparatus (EscN) is essential for intestinal colonization; however, the type III secreted effector protein Map plays only a minor role . Other Type III secretion-associated genes were implicated in colonization of calves by E . coli O157 : H7, including z0990 (ecs0850), which encodes the non-LEE-encoded type III secreted effector NleD and the closely related z3023 (ecs2672) and z3026 (ecs2674) genes which encode homologues of Shigella IpaH proteins . We also identified a novel fimbrial locus required for intestinal colonization in calves by E . coli O157 : H7 (z2199-z2206; ecs2114-ecs2107/locus 8) and demonstrated that a mutant harbouring a deletion of the putative major fimbrial subunit gene is rapidly out-competed by the parent strain in co-infection studies . Our data provide valuable new information for the development of intervention strategies.

Mol Microbiol, 2004 Nov, 54(4), 1117 - 33
Direct and indirect transcriptional activation of virulence genes by an AraC-like protein, PerA from enteropathogenic Escherichia coli; Porter ME et al.; The plasmid-encoded Per regulatory locus of enteropathogenic Escherichia coli (EPEC) is generally considered to consist of three genes, perA, perB and perC . PerA, a member of the AraC-like family of transcriptional regulators, is known to be an activator of its own promoter (autoactivation) as well as of the plasmid-located bfp operon encoding bundle-forming pili, but its role in activation of the chromosomal locus of enterocyte effacement (LEE) pathogenicity island, which confers the property of intimate adherence on EPEC, requires clarification . Here, we show that PerA is also required for activation of the master regulatory LEE operon, LEE1, but that this activation is indirect, being achieved via autoactivation of the per promoter which ensures sufficient production of the PerC protein to activate LEE1 . In contrast, PerA-dependent activation of the per and bfp promoters is direct and does not require the other Per proteins, but is modulated by the nucleoid-associated protein H-NS . The closely related VirF regulator from Shigella flexneri cannot substitute for PerA to activate these promoters, despite being able to bind their upstream regions in vitro . PerA can bind the per and bfp promoter fragments to form multiple complexes, while VirF forms only a single complex . Site-directed mutagenesis of the PerA protein suggests that, like VirF, it may use both of its carboxy-terminal helix-turn-helix motifs for DNA interaction, and may also make direct contacts with RNA polymerase . In addition, we have isolated mutations in the poorly characterized amino-terminal domain of PerA which affect its ability to activate gene expression.

Clin Microbiol Infect, 2004 Nov, 10(11), 1033 - 5
Increasing incidence of resistance to nalidixic acid in shigellas from humans in England and Wales: implications for therapy; Cheasty T et al.; Among shigellas isolated from patients in England and Wales in 2002, 10% of subgroups A, B and C, and 13% of subgroup D (Shigella sonnei), were resistant to nalidixic acid . As a consequence, should antimicrobial therapy be indicated, the efficacy of nalidixic acid as the preferred treatment for children with bacillary dysentery has been jeopardised.

J Bacteriol, 2004 Nov, 186(22), 7773 - 82
Single-molecule approach to bacterial genomic comparisons via optical mapping; Zhou S et al.; Modern comparative genomics has been established, in part, by the sequencing and annotation of a broad range of microbial species . To gain further insights, new sequencing efforts are now dealing with the variety of strains or isolates that gives a species definition and range; however, this number vastly outstrips our ability to sequence them . Given the availability of a large number of microbial species, new whole genome approaches must be developed to fully leverage this information at the level of strain diversity that maximize discovery . Here, we describe how optical mapping, a single-molecule system, was used to identify and annotate chromosomal alterations between bacterial strains represented by several species . Since whole-genome optical maps are ordered restriction maps, sequenced strains of Shigella flexneri serotype 2a (2457T and 301), Yersinia pestis (CO 92 and KIM), and Escherichia coli were aligned as maps to identify regions of homology and to further characterize them as possible insertions, deletions, inversions, or translocations . Importantly, an unsequenced Shigella flexneri strain (serotype Y strain AMC{328Y}) was optically mapped and aligned with two sequenced ones to reveal one novel locus implicated in serotype conversion and several other loci containing insertion sequence elements or phage-related gene insertions . Our results suggest that genomic rearrangements and chromosomal breakpoints are readily identified and annotated against a prototypic sequenced strain by using the tools of optical mapping.

Antimicrob Agents Chemother, 2004 Nov, 48(11), 4250 - 5
Complex multiple antibiotic and mercury resistance region derived from the r-det of NR1 (R100); Partridge SR et al.; The sequence of the 45.2-kb multidrug and mercury resistance region of pRMH760, a large plasmid from a clinical isolate of Klebsiella pneumoniae collected in 1997 in Australia, was completed . Most of the modules found in the resistance determinant (r-det), or Tn2670, region of NR1 (also known as R100), isolated from a Shigella flexneri strain in Japan in the late 1950s, were present in pRMH760 but in a different configuration . The location was also different, with the Tn2670-derived region flanked by the transposition module of Tn1696 and a mercury resistance module almost identical to one found in the plasmid pDU1358 . This arrangement is consistent with a three-step process . First, the r-det was circularized via homologous recombination between the IS1 elements and reincorporated at a new location, possibly in a different plasmid, via homologous recombination between the 5'-conserved (5'-CS) or 3'-CS of the In34 integron in the r-det and the same region of a second class 1 integron in a Tn1696 relative . Subsequently, resolvase-mediated recombination between the res sites in the r-det and a second mercury resistance transposon removed one end of the Tn1696-like transposon and part of the second transposon . Other events occurring within the r-det-derived portion have also contributed to the formation of the pRMH760 resistance region . Tn2 or a close relative that includes the bla(TEM-1b) gene had moved into the Tn21 mercury resistance module with subsequent deletion of the adjacent sequence, and all four 38-bp inverted repeats corresponding to Tn21 family transposon termini have been interrupted by an IS4321-like element.

J Med Microbiol, 2004 Nov, 53(Pt 11), 1075 - 81
Role of anti-CD3 in modulation of Th1-type immune response in Shigella dysenteriae infection; Sinha AK et al.; A murine model was used to evaluate the role of anti-CD3 in modulating a Th1-type response by restimulation of T-cells after immunization with the 57 kDa immunodominant antigen of Shigella dysenteriae 1 outer-membrane proteins (OMPs), followed by Shigella infection after immunization . To observe the effect of anti-CD3, other T-cell cultures were also established following anti-CD1, anti-IL2 and phytohaemagglutinin stimulation . Anti-CD3 stimulation of reconstituted T-cells showed 'mean' levels of CD4 and CD25 were enhanced by 34.5 and 31.1 % in immunized mice, which was comparable to 53.2 and 50.7 %, respectively, in challenged-immunized mice, and were dominant over CD8+ T-cells . Levels of IL2 generated by anti-CD3-stimulated T-cells of immunized mice were greater than those of unstimulated T-cells and were significantly elevated in challenged-immunized mice . The reactivity of T-cells indicated their complete responsiveness, as anti-CD3 antibody might not inhibit the migration of the macrophages but rather inhibit IL4 . These macrophage factors synergistically act with anions towards an activated response, which in turn provokes IL2 secretion with a low degree of internalization of its receptor . Thus, sharing of IL2 to form a high-affinity receptor complex with CD4+ T-cells through motive signals suggested a generalized T-cell activation with increased humoral responses . Macrophage migration inhibition factor (MIF) and IL4 responses during anti-CD3 stimulation of immunized mice indicated that the role of anti-CD3 in generation of O2- is due to a synergistic effect by Th1 subsets of Th0 cells . The above findings should have implications for understanding the immunoregulatory role of anti-CD3 associated with 57 kDa antigen in immunoprophylactic measures.

J AOAC Int, 2004 Sep-Oct, 87(5), 1115 - 22
Alternative anaerobic enrichments to the bacteriological analytical manual culture method for isolation of Shigella sonnei from selected types of fresh produce; Jacobson AP et al.; Alternative methods of reducing oxygen during anaerobic enrichment in the Bacteriological Analytical Manual (BAM) Shigella culture method were evaluated and compared to the current and less practical GasPak method . The alternative anaerobic methods included the use of reducing agents in Shigella broth and reducing culture container headspace volume to minimize atmospheric effects on oxygen concentration in Shigella broth during enrichment . The reducing agents evaluated were sodium thioglycollate, L-cystine, L-cysteine, titanium(III) citrate, and dithiothreitol, each at concentrations of 0.1, 0.05, and 0.01% . The use of Oxyrase for Broth with the enrichment medium (Shigella broth) was evaluated at concentrations of 10, 20 and 30 microL/mL . Recoveries of chill- and freeze-stressed S . sonnei strains 357 and 20143 were determined with each anaerobic method, including the GasPak method, using inoculation levels ranging from 10(0)to 10(3) cells . For each anaerobic method, strain, inoculation level, and stress type, 5 replicate enrichments were evaluated by streaking to MacConkey agar for isolation . The numbers of cultures with each method from which S . sonnei was isolated were used to compare the alternative anaerobic methods to the GasPak method . The alternative anaerobic method with which chill- and freeze-stressed S . sonnei strains 357 and 20143 were isolated most consistently was the use of Oxyrase for Broth in Shigella broth at a concentration of 20 microL/mL . This method was compared to the GasPak anaerobic method in evaluations on the recovery of S . sonnei strains 357 and 20143 from artificially contaminated test portions of parsley, cilantro, green onions, strawberries, carrots, and celery . A third anaerobic method included the use of 0.5 cm mineral oil overlay on cultures containing Oxyrase for Broth at concentrations of 20 microL/mL . Recovery rates of strain 357 were significantly greater (p < 0.05) with the GasPak method than with Oxyrase for Broth, with and without the 0.5 cm mineral oil overlay, for test portions of parsley, cilantro, and celery . When Oxyrase for Broth was used with Shigella broth, strain 357 was isolated at higher rates from all produce types, except cilantro, when 0.5 cm mineral oil overlay was applied to enrichment cultures . The use of mineral oil overlay with Oxyrase for Broth also improved recovery of strain 20143 from test portions of all produce types except green onion and strawberries . These differences were significant (p < 0.05) with parsley, carrots, and cilantro (1 of 2 evaluations) . No statistically significant differences (p > 0.05) between the GasPak and Oxyrase for Broth anaerobic methods occurred when mineral oil overlay was used with Oxyrase for Broth . The use of Oxyrase for Broth with a 0.5 cm mineral oil overlay is a practical alternative for anaerobic enrichment with the BAM method in the analysis of some produce types . Differences in recovery among the different produce types and methods occurred between S . sonnei strains 357 and 20143, emphasizing the need for additional S . sonnei strains in future evaluations.

Afr J Med Med Sci, 2004 Mar, 33(1), 51 - 5
Substrate profile variation and drug resistance patterns of â - lactamase producing Shigella species isolated from diarrhoeal patients in Lagos, Nigeria; Iwalokur BA et al.; The number and trend of antibiotic resistance by Shigella species recovered from food and diarrhoeal stools are on the increase in Nigeria and has resulted in a high frequency of hospitalisation . Increased cost of disease management, and higher mortality in children . This study exposes 51 a-lactamase producing Shigella isolates from Lagos to some newly introduced drugs in the country . The drugs include a-lactam - a-actamase inhibitor antibiotics . a-lctam substrate hydrolysis and inhibitory effects of clavulanate were also investigated in-vitro . Results obtained revealed that all the isolates showed high level resistance to tetracycline, ampicillin, streptomycin, co-trimoxazole and amoxicillin with an MIC range of 128 - 1024 microg/ml . The isolates were susceptible to piperacillin, tobramycin, aztreonam and ofloxacin (0.03 - 8 microg/ ml) . 18.2 - 40.9% of S . flexneri and S . dysenteriae showed low level resistance to cefuroxime and cefotaxime (MIC = 4 - 16 microg/ ml) . Among the a-lactam - a- lctamase inhibitors tested, only piperacillin-tazobactam showed 100% resistance . Hydrolysis of a -lctam substrate was found to be species dependent in decreasing order of S . flexneri, S . dysenteriae, S . sonnei and S.boydii . An IC50 range of 0.8-2.4 mM was also observed in these isolates . Our data indicate that the incidence of multidrug resistance is high among a-lactamase producing Shigella isolates in Lagos, Nigeria . While the third generation cephalosporins should be used with cautions, some of the newly introduced drugs have the prospects of being used in the future control and management of shigellosis in the country.

J Bacteriol, 2004 Nov, 186(21), 7460 - 5
Molecular basis of the indole-negative reaction in Shigella strains: extensive damages to the tna operon by insertion sequences; Rezwan F et al.; The molecular basis of the loss of tryptophan utilization (indole-negative phenotype) of Shigella strains, in effect clones of Escherichia coli, was investigated . Analysis of the tna operon of 23 Shigella strains representing each of the indole-negative serotypes revealed that insertion sequence-mediated insertion and/or deletions damaged the tna operon, leading to inability to convert tryptophan to indole . These events differ for cluster 1, cluster 3, and the outlier Shigella strains, confirming our previous observation of independent origins of these lineages from within E . coli . Parallel loss of the trait and prevalence of indole-negative strains suggest that the trait is deleterious in Shigella strains and advantages those without it.

Yi Chuan Xue Bao, 2004 Apr, 31(4), 369 - 74
{Identification of a protein interacting with Shigella flexneri IpaC invasin by yeast two-hybrid system}; Yan XY et al.; Two-hybrid system was applied to screen proteins interacting with IpaC in the host cell . By using two-hybrid system, the bait plasmid containing ipaC gene was constructed and designated pGBKT-IpaC . A human HeLa cDNA library was screened to isolate protein factors that might interact with IpaC . Among the 2 x 10(6) clones screened, 22 positive clones were picked out . Sequence analysis revealed that two of them contained cDNA fragments from collagenase . Subsequently the domain of IpaC interacting with collagenase fragment was identified . These results suggest that IpaC might play a role in some biological processes where collagenase is involved.

Mol Immunol, 2004 Nov, 41(12), 1167 - 75
Up-regulation of CD80-CD86 and IgA on mouse peritoneal B-1 cells by porin of Shigella dysenteriae is Toll-like receptors 2 and 6 dependent; Ray A et al.; Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptor (TLR) 2 and TLR6 by 1.5- and 2.9-fold respectively, of peritoneal cavity B-1a and B-1b cells, implicating that coexpression of TLR2 and TLR6 is essential as a combinatorial repertoire for recognition of porin by the B-1 cells . Among the two key TLRs, TLR2 and TLR4, which are primarily responsible for recognizing majority of the bacterial products, TLR2 and not TLR4, participates in porin recognition . TLR2 got increased on both the B-1 cell populations whereas the TLR4 expression remained unaffected . Besides TLRs, mRNA for MyD88, an effector molecule associated with TLR-mediated response was enhanced by 1.8-fold that suggests of its involvement in the activity of porin . Both of the B-1 cell populations expressed strongly the mRNA for NF-kappaB in the presence of porin, that was 2.4-fold more than untreated control, conforming to the earlier finding that coexpression of TLR2 and TLR6, resulted in robust NF-kappaB activation for signaling . Porin treatment of B-1 cell populations of C57BL/6 mice, and C3H/HeJ mice in particular, selectively up-regulated the expression of the costimulatory molecules . CD80 expression got enhanced on the B-1a cells whereas CD86 got solely expressed on B-1b cells . Porin-induced cell surface expression of IgM and IgA on B-1 cell populations from C57BL/6 mice . The IgA-generating capacity, hallmark of mucosal immune response, was confirmed with B-1 cells of C3H/HeJ, the lipopolysaccharide non-responder mouse, in response to the protein . The porin-mediated induction of IgA was augmented by interleukin-6 on B-1a and B-1b cells, by 2.4- and 2.6-fold, respectively . The IgA expressed on both B-1a and B-1b cell surfaces after 72 h of culture was found to bind to the 38 kDa monomer of porin confirming it to be anti-porin IgA antibody.

Genome Res, 2004 Nov, 14(11), 2273 - 8 Epub 2004 Oct 12.
Psi-Phi: exploring the outer limits of bacterial pseudogenes; Lerat E et al.; Because bacterial chromosomes are tightly packed with genes and were traditionally viewed as being optimized for size and replication speed, it was not surprising that the early annotations of sequenced bacterial genomes reported few, if any, pseudogenes . But because pseudogenes are generally recognized by comparisons with their functional counterparts, as more genome sequences accumulated, many bacterial pathogens were found to harbor large numbers of truncated, inactivated, and degraded genes . Because the mutational events that inactivate genes occur continuously in all genomes, we investigated whether the rarity of pseudogenes in some bacteria was attributable to properties inherent to the organism or to the failure to recognize pseudogenes . By developing a program suite (called Psi-Phi, for Psi-gene Finder) that applies a comparative method to identify pseudogenes (attributable both to misannotation and to nonrecognition), we analyzed the pseudogene inventories in the sequenced members of the Escherichia coli/Shigella clade . This approach recovered hundreds of previously unrecognized pseudogenes and showed that pseudogenes are a regular feature of bacterial genomes, even in those whose original annotations registered no truncated or otherwise inactivated genes . In Shigella flexneri 2a, large proportions of pseudogenes are generated by nonsense mutations and IS element insertions, events that seldom produce the pseudogenes present in the other genomes examined . Almost all (>95%) pseudogenes are restricted to only one of the genomes and are of relatively recent origin, suggesting that these bacteria possess active mechanisms to eliminate nonfunctional genes.

J Health Popul Nutr, 2004 Jun, 22(2), 170 - 81
The health belief model and factors relating to potential use of a vaccine for shigellosis in Kaeng Koi district, Saraburi province, Thailand; Butraporn P et al.; Shigellosis is an important cause of morbidity and mortality throughout the world . Approximately, 1.1 million deaths occur a year due to this disease, making it the fourth leading cause of mortality worldwide . This paper explores local interest in and potential use of a vaccine for shigellosis in Thailand where Shigella poses an important public-health concern . Data for this study were collected during June-November 2002 from 522 subjects surveyed using a sociobehavioural questionnaire in Kaeng Koi district in central Thailand . The community demand and likely use of a vaccine were examined in relation to the Health Belief Model, which provides analytical constructs for investigating the multiple issues of local readiness to accept and access a new vaccine . As the key outcome variable, most respondents showed interest in receiving a vaccine against dysentery which they thought would provide useful protection against the disease . However, there was only a moderate number who perceived dysentery as serious and themselves as susceptible to it, although it was perceived to cause some burden to and additional expense for families . Most people identified a number of groups who were thought to be especially vulnerable to dysentery, such as the elderly, pre-school, and school-age children, and poor labourers . Other outcomes of the study included the identification of acceptable and convenient sites for its delivery, such as government health clinics and private clinics, and respected sources for information about the vaccine, such as health clinic personnel and community health volunteers . This information suggests that components of the Health Belief Model may be useful in identifying community acceptance of a vaccine and the means of introducing it . This health information is important for planning and implementing vaccine programmes.

J Health Popul Nutr, 2004 Jun, 22(2), 150 - 8
Acceptability and accessibility of a Shigellosis vaccine in Nha Trang city of Viet Nam; Kaljee LM et al.; The acceptability and accessibility of a hypothetical Shigella vaccination campaign was explored . A household survey was conducted with 539 randomly-selected residents of six communes in Nha Trang city of Viet Nam . Four categories of acceptability, such as refusers, low acceptors, acceptors, and high acceptors, were established, Refusers were significantly more likely to be elderly women and were less likely to know the purpose of vaccinations . Low acceptors tended to be male, elderly, and live in urban areas . Low acceptors perceived the disease as less serious and themselves as less vulnerable than acceptors and high acceptors . In terms of accessing vaccination, the commune health centre workers and commune leaders were the preferred sources of information and commune health centres the preferred location for vaccination . Direct verbal information from healthcare providers and audio-visual media were preferred to written information . The respondents expressed a desire for knowledge about the side-effects and efficacy of the vaccine . These findings are significant for targeting specific messages about shigellosis and vaccination to different populations and maximizing informed participation in public-health campaigns.

Commun Dis Intell, 2004, 28(2), 225 - 9
An outbreak of shigellosis in a child care centre; Genobile D et al.; Outbreaks of shigellosis in child care are not commonly reported in Australia, however Shigella bacteria can easily spread in these settings . We report an outbreak of shigellosis in a child care centre and discuss the control measures implemented . This investigation identified 20 confirmed cases of Shigella sonnei biotype g and a further 47 probable cases in children and staff who attended a child care centre, and their household contacts . The investigation highlighted the importance of stringent control measures and protocols for dealing with outbreaks of Shigella and other enteric infections in the child care setting, and the importance of prompt notification by both doctors and child care centres, of suspected outbreaks.

Bull Exp Biol Med, 2004 May, 137(5), 479 - 82
Invasive characteristics of apathogenic Shigella flexneri 5a2c mutant obtained under the effect of furazolidone; Efremova TN et al.; Apathogenic Shigella flexneri 5a2c mutant treated with furazolidone can infect eucaryotic cells . These bacteria contain no virulence genes responsible for Sh . flexneri invasion, which seems to be the cause of their apathogenicity . The capacity of bacteria to penetrate into eucaryotic cells correlates with the appearance of ECP 32 protease specifically cleaving actin.

Am J Trop Med Hyg, 2004 Sep, 71(3), 367 - 72
Epidemiology of Shigella-associated diarrhea in rural Egyptian children; Abu-Elyazeed RR et al.; During the period from February 1995 to February 1998, the epidemiology of Shigella diarrhea was studied among children less than three years of age residing in Egypt's Nile Delta . Children were visited twice a week and a stool sample was collected from any of them with diarrhea . The incidence of Shigella-associated diarrhea was 0.2 episodes/child-year, with S . flexneri being the most common serogroup isolated (55% of Shigella episodes) . Younger age and the warm months increased the risk of developing Shigella-associated diarrhea, while breastfeeding was protective . Children with Shigella were ill for a mean of four days and passed a mean of six stools per day . Common symptoms included fever (35%), vomiting (19%), and dehydration (16%) . Dysentery, however, was unusual, occurring in only 11% of the cases . In conclusion, Shigella-associated diarrhea remains relatively common in Egyptian children and supports the need for additional control measures including vaccine development.

J Biol Chem, 2004 Nov 12, 279(46), 47792 - 8 Epub 2004 Sep 10.
Rafts can trigger contact-mediated secretion of bacterial effectors via a lipid-based mechanism; van der Goot FG et al.; Infection by the Gram-negative bacterial pathogen Shigella flexneri depends on its ability to invade host cells . Bacterial engulfment requires a functional type III secretion system (TTSS) allowing the translocation into host cells of bacterial effectors that activate cell-signaling cascades . We demonstrated previously that specialized lipid membrane domains enriched in cholesterol and sphingolipids (rafts) are involved during early steps of invasion, namely in binding and host cell entry . In this study, we addressed the issue of contact-mediated secretion by the TTSS . We show that contact-mediated and TTSS-induced hemolysis depend on the presence of cholesterol on the host cell surface . We found that purified detergent resistant membranes were able to activate TTSS . Finally, we found that artificial liposomes, devoid of proteins, were able to activate the TTSS but only when their composition mimicked that of lipid rafts . Altogether, these data indicate that specific lipid packing can trigger contact-mediated secretion by S . flexneri.

FEMS Immunol Med Microbiol, 2004 Oct 1, 42(2), 255 - 9
Polymicrobial infections in children with diarrhoea in a rural area of Jordan; Nimri LF et al.; Polymicrobial infections associated with diarrhoea are common in developing countries . Stool specimens were collected from 220 patient children and 100 controls . Potential pathogenic agents isolated from 143 (65%) children were identified by molecular and standard microbiological methods . Co-infections with two or more agents were detected in 50 (35%) cases . Escherichia coli, Shigella dysenteriae, Giardia and Entamoeba histolytica were found to be predominant . The etiologic agents could not be determined in 77 (35%) cases . The most significant risk factors were the age, the education level of the mother and the use of non-chlorinated water . The high infection rate of diarrhoeal diseases is a strong indication that these pathogens circulate easily through the population.

Trans R Soc Trop Med Hyg, 2004 Nov, 98(11), 635 - 43
Case management of a multidrug-resistant Shigella dysenteriae serotype 1 outbreak in a crisis context in Sierra Leone, 1999-2000; Guerin PJ et al.; From December 1999 to the end of February 2000, 4218 cases of dysentery were reported in Kenema district, southeastern Sierra Leone, by a Medecins Sans Frontieres team operating in this region . Shigella dysenteriae serotype 1 was isolated from the early cases . The overall attack rate was 7.5% but higher among children under 5 years (11.2%) compared to the rest of the population (6.8%) (RR = 1.6; 95% CI 1.5-1.8) . The case fatality ratio was 3.1%, and higher for children under 5 years (6.1% vs . 2.1%) (RR = 2.9; 95% CI 2.1-4.1) . A case management strategy based on stratification of affected cases was chosen in this resource-poor setting . Patients considered at higher risk of death were treated with a 5 day ciprofloxacin regimen in isolation centres . Five hundred and eighty-three cases were treated with a case fatality ratio of 0.9% . Patients who did not have signs of severity when seen by health workers were given hygiene advice and oral rehydration salts . This strategy was effective in this complex emergency.

J Immunol, 2004 Sep 15, 173(6), 4197 - 206
IL-8 is a key chemokine regulating neutrophil recruitment in a new mouse model of Shigella-induced colitis; Singer M et al.; The lack of a mouse model of acute rectocolitis mimicking human bacillary dysentery in the presence of invasive Shigella is a major handicap to study the pathogenesis of the disease and to develop a Shigella vaccine . The inability of the mouse intestinal mucosa to elicit an inflammatory infiltrate composed primarily of polymorphonuclear leukocytes (PMN) may be due to a defect in epithelial invasion, in the sensing of invading bacteria, or in the effector mechanisms that recruit the PMN infiltrate . We demonstrate that the BALB/cJ mouse colonic epithelium not only can be invaded by Shigella, but also elicits an inflammatory infiltrate that, however, lacks PMN . This observation points to a major defect of mice in effector mechanisms, particularly the lack of expression of the CXC chemokine, IL-8 . Indeed, this work demonstrates that the delivery of recombinant human IL-8, together with Shigella infection of the colonic epithelial surface, causes an acute colitis characterized by a strong PMN infiltrate that, by all criteria, including transcription profiles of key mediators of the innate/inflammatory response and histopathological lesions, mimics bacillary dysentery . This is a major step forward in the development of a murine model of bacillary dysentery .

Clin Microbiol Infect, 2004 Sep, 10(9), 804 - 10
Antimicrobial susceptibility trends among Escherichia coli and Shigella spp . isolated from rural Egyptian paediatric populations with diarrhoea between 1995 and 2000; Putnam SD et al.; Antimicrobial susceptibility testing was performed on 3,627 isolates of Escherichia coli and 180 isolates of Shigella spp . collected in rural locations from 875 Egyptian children with diarrhoea between 1995 and 2000 . The cumulative rates of resistance for E . coli and Shigella spp . were high (respectively, 68.2% and 54.8% for ampicillin, 24.2% and 23.5% for ampicillin-sulbactam, 57.2% and 42.5% for trimethoprim-sulphamethoxazole, and 50.9% and 75.4% for tetracycline) . Non-enterotoxigenic E . coli (NETEC) isolates had a consistently higher level of antimicrobial resistance than did enterotoxigenic E . coli (ETEC) isolates . Trend testing showed significant decreases in resistance to ampicillin, ampicillin-sulbactam and tetracycline among all E . coli isolates . Increasing rates of resistance were observed for trimethoprim-sulphamethoxazole in ETEC isolates and Shigella spp., but not in NETEC isolates . Low levels of resistance were observed for all other antimicrobial agents tested . Overall, high levels, but decreasing trends, of resistance to commonly used antimicrobial agents were detected among isolates of E . coli and Shigella spp . from children in rural Egypt.

J Antimicrob Chemother, 2004 Oct, 54(4), 730 - 4 Epub 2004 Sep 03.
Genetic relatedness of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated in south Asia; Talukder KA et al.; OBJECTIVES: The aim of the present study was to determine the clonal relationships of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated from south Asia, and S . dysenteriae 1 strains associated with epidemics in 1978, 1984 and 1994 . METHODS: The antimicrobial susceptibilities were examined by NCCLS methods . Molecular epidemiological characterization was performed by plasmid profiling, pulsed-field gel electrophoresis (PFGE) and mutation analysis of the quinolone resistance-determining region (QRDR) of gyrA by sequencing . RESULTS: Plasmid patterns of the current ciprofloxacin-resistant strains from India, Nepal and Bangladesh were very similar to those of the 1978, 1984 and 1994 epidemic isolates of S . dysenteriae 1, except for the presence of a new plasmid of approximately 2.6 MDa, which was found in one recent ciprofloxacin-resistant strain isolated in Bangladesh . PFGE analysis showed that the ciprofloxacin-resistant strains isolated in Bangladesh, India and Nepal belonged to a PFGE type (type A), which was possibly related to that of the 1984 and 1994 clone of S . dysenteriae 1, but different from 1978 epidemic strains . The current ciprofloxacin-resistant strains belong to five subtypes (A3-A7), all of which were found in India, but in Bangladesh and Nepal, only A3 existed . Mutation analysis of the QRDR of gyrA revealed that amino acid substitutions at positions 83 and 87 of ciprofloxacin-resistant strains isolated in Bangladesh were similar to those of the strains isolated in Nepal, but different (at position 87) from ciprofloxacin-resistant strains isolated in India . CONCLUSIONS: PFGE and mutation analysis of gyrA showed differences between the current ciprofloxacin-resistant S . dysenteriae 1 strains isolated in south Asia and those associated with epidemics in 1978, 1984 and 1994.

Infect Immun, 2004 Sep, 72(9), 5080 - 8
Molecular evolutionary relationships of enteroinvasive Escherichia coli and Shigella spp; Lan R et al.; Enteroinvasive Escherichia coli (EIEC), a distinctive pathogenic form of E . coli causing dysentery, is similar in many properties to bacteria placed in the four species of Shigella . Shigella has been separated as a genus but in fact comprises several clones of E . coli . The evolutionary relationships of 32 EIEC strains of 12 serotypes have been determined by sequencing of four housekeeping genes and two plasmid genes which were used previously to determine the relationships of Shigella strains . The EIEC strains were grouped in four clusters with one outlier strain, indicating independent derivation of EIEC several times . Three of the four clusters contain more than one O antigen type . One EIEC strain (an O112ac:H- strain) was found in Shigella cluster 3 but is not identical to the Shigella cluster 3 D2 and B15 strains with the same O antigen . Two forms of the virulence plasmid pINV have been identified in Shigella strains by using the sequences of ipgD and mxiA genes, and all but two of our EIEC strains have pINV A . The EIEC strains were grouped in two subclusters with a very low level of variation, generally not intermingled with Shigella pINV A strains . The EIEC clusters based on housekeeping genes were reflected in the plasmid gene sequences, with some exceptions . Two strains were found in the pINV B form by using the ipgD sequence, with one strain having an mxiA sequence similar to the divergent sequence of D1 . Clearly, EIEC and Shigella spp . form a pathovar of E . coli.

Epidemiol Infect, 2004 Aug, 132(4), 773 - 7
Shigella serotypes among hospitalized patients in urban Bangladesh and their antimicrobial resistance; Khan AI et al.; We studied the isolation of Shigella spp., and their antimicrobial resistance . S . flexneri (54 %) was most frequently isolated, followed by S . dysenteriae (20 %), S . boydii (16 %) and S . sonnei (10 %) . Among S . flexneri (n = 122), 29 (24 %) were 2a, and 23 (19 %) were 2b . None of the Shigella strains were resistant to mecillinam or ciprofloxacin . Resistance to nalidixic acid was most frequent among S . dysenteriae type 1 (100%) followed by S . flexneri 2a (69%), and S . flexneri 2b (52 %) . Systematic monitoring is needed to identify most prevalent serotypes, and to detect changes in the prevalence and antimicrobial resistance pattern.

Epidemiol Infect, 2004 Aug, 132(4), 579 - 83
Epidemiological features of a newly described serotype of Shigella boydii; Kalluri P et al.; We report the clinical, microbiological, and epidemiological features of an emerging serotype, Shigella boydii 20 . We interviewed patients about symptoms, and history of travel and visitors during the week before illness onset . Seventy-five per cent of the 56 patients were Hispanic . During the week before illness onset, 18 (32%) travelled abroad; 17 (94%) had visited Mexico . Eight (21%) out of 38 who had not travelled had foreign visitors . There were eight closely related patterns by PFGE with XbaI . S . boydii 20 may be related to travel to Mexico and Hispanic ethnicity . Prompt epidemiological investigation of clusters of S . boydii 20 infection may help identify specific vehicles and risk factors for infection.

Int J STD AIDS, 2004 Aug, 15(8), 533 - 7
Shigellosis - a re-emerging sexually transmitted infection: outbreak in men having sex with men in Berlin; Marcus U et al.; In December 2001, the Robert Koch-Institut (RKI) was informed about a cluster of 10 Shigella sonnei infections in men who have sex with men (MSM), diagnosed in Berlin since September 2001 . A retrospective investigation on sexual risk factors for infection in all shigella cases from Berlin from 2001 was initiated by sending a questionnaire to all patients without known travel history . Simultaneously laboratories were asked to send new shigella isolates from patients to the National Reference Centre at the RKI . Out of 29 responders, 24 self-identified as MSM . Seventeen of these reported sexual contacts as the most likely potential risk of infection . Almost all MSM reported direct or indirect oral-anal sexual contacts in the week before they fell ill . Fifteen of 27 shigella isolates collected during the prospective sampling period showed identical plasmid profiles, PFGE- and resistance patterns and can therefore be regarded as clonally identical . Asymptomatic and/or prolonged shedding in the reconvalescent phase may contribute to the transmission risk of shigella infection during oral-anal sexual practices . Awareness among practitioners and among MSM about the risk of sexual transmission of orally transmitted agents needs to be raised.

Zhong Yao Cai, 2004 Apr, 27(4), 270 - 2
{Studies on antibacterial activities of secondary metabolites from fungus Cephalosporium sp . AL031}; Yunmei B et al.; The antibacterial activity of fourteen identified compounds from secondary metabolites of fungus Cephalosporium sp . AL031 against eleven gram-positive or gram-negative bacteria in vitro was investigated by agar diffusion assay . The results suggested that two new compounds {S-(+)-5-hydroxymellein, S-(+)-7-hydroxymellein}, together with four known compounds {S-(+)-mellein, 2,4-dihydroxy-3,6-dimethylbenzoic acid, 3,6-dihydroxy-2,4-dimethylbenzoic acid and succinic acid} had broad spectrum antibacterial activity . The minimum inhibitory concentrations (MIC) of these six compounds against Shigella sonnei, Mycobacterium tuberculosis and Streptococcus pneumoniae were determined . Their MIC were 0.03125 to approximately 0.5 mg/ml.

J Clin Microbiol, 2004 Aug, 42(8), 3799 - 804
Identification of Escherichia coli O114 O-antigen gene cluster and development of an O114 serogroup-specific PCR assay; Feng L et al.; Screening for the Escherichia coli O serotype is the traditional test for identification of E . coli clones . The O-antigen gene cluster of the E . coli O114 type strain was sequenced, and 12 open reading frames were assigned functions on the basis of homology . By screening against all 186 E . coli and Shigella O serotypes, five genes specific to E . coli O114 were identified . A PCR assay based on the O-antigen-specific genes was developed and tested on 41 clinical isolates of E . coli O114 . The PCR assay was shown to be highly specific and sensitive . When tested with pork and water samples, as few as 0.12 CFU of E . coli O114 g(-1) were detected . Thus, the PCR assays established in this study can be used to reliably identify E . coli O114 strains and may also be used to detect E . coli O114 strains in food, water, and other environmental samples.

Am J Infect Control, 2004 Aug, 32(5), 274 - 7
Is there an association between shigellosis incidence and socioeconomic status in metropolitan Haifa?
Dubnov J, Rubin L, Rishpon S.
BACKGROUND: Shigellosis incidence rates in Israel have declined continuously over the past 50 years, but they remain 20 times greater than those in the United States . Socioeconomic factors may influence shigellosis morbidity, but this may be difficult to demonstrate in the absence of data for individual patients and when using composite rates for large geographic areas . Use of census tract data for small, relatively homogeneous geographic areas may lessen the effects of the "ecological fallacy." The present study analyzes the effect of socioeconomic status (SES) on shigellosis morbidity in the Haifa metropolitan region . METHODS: The study population consisted of the 7 cities in the Haifa subdistrict that constitute the greater metropolitan region . Cases of shigellosis reported during the years 2000 and 2001 were mapped, and age-standardized rates were calculated for the census tract areas . The incidence rates were then compared with the SES category of the census tract using the Kruskal-Wallis test . RESULTS: No association was found between incidence rates of shigellosis and SES category of the census tract areas in the Haifa metropolitan area for the years 2000 and 2001 (Kruskal-Wallis chi(2)=0.440; P=.803) . CONCLUSION: We found no association between shigellosis morbidity and socioeconomic status . This finding is probably real and not the result of reporting bias . Analysis of morbidity using small geographical units such as census tracts is more accurate than analysis using large geographical areas such as cities.

J Bacteriol, 2004 Aug, 186(16), 5551 - 4
Excision of the Shigella resistance locus pathogenicity island in Shigella flexneri is stimulated by a member of a new subgroup of recombination directionality factors; Luck SN et al.; Pathogenicity islands are capable of excision and insertion within bacterial chromosomes . We describe a protein, Rox, that stimulates excision of the Shigella resistance locus pathogenicity island in Shigella flexneri . Sequence analysis suggests that Rox belongs to a new subfamily of recombination directionality factors, which includes proteins from P4, enterohemorrhagic Escherichia coli, and Yersinia pestis.

Southeast Asian J Trop Med Public Health, 2004 Mar, 35(1), 181 - 7
Antimicrobial use in children under five years with diarrhea in a central region province, Thailand; Howteerakul N et al.; This cross-sectional study aimed to estimate the prevalence of appropriate antimicrobial prescribing for treating childhood diarrhea within the public hospital system in a central region province, Thailand . Reported are findings of a prospective clinical audit of 424 cases treated by 38 physicians . Appropriate use of antimicrobials was defined as prescribing antimicrobials for managing an invasive bacterial-type, bloody diarrhea or not prescribing antimicrobials for managing a watery-type or non-bloody diarrhea . Among 424 cases with diarrhea, 12.5% were invasive bacterial-type . Of the 66 diarrheal episodes in which stool samples were cultured, 7 stool specimens were positive, two with Shigella sonnei, two with Vibrio cholerae Ogawa and three with E . coli . Based on the presence of mucus and blood in stools, 27.4% of 424 cases received appropriate antimicrobial drugs . Cotrimoxazole was the most commonly prescribed drug (51%), followed by colistin sulfate (15.3%), norfloxacin (11%), and nalidixic acid (0.5%) . The average number of antimicrobials per case of inpatients was higher than outpatients (1.15 vs 0.84, p < 0.001) . There was a trend toward prescribing norfloxacin in childhood diarrhea . The Ministry of Public Health should continue providing effective interventions aimed at improving physicians' knowledge of diarrhea treatment . Similar efforts should be directed toward improving caretakers' knowledge about home care for childhood diarrhea and encouraging widespread correct use of oral rehydration therapy (ORT) in the community . Hopefully, such activities will help reduce the inappropriate use of antimicrobial agents in treating diarrheal disease.

Southeast Asian J Trop Med Public Health, 2004 Mar, 35(1), 97 - 108
Perceptions of Shigella and of Shigella vaccine among rural Chinese: compatibility with Western models of behavioral change; Youlong G et al.; Shigella remain a major source of morbidity and mortality in developing countries, including China . In response, national and international researchers are actively working to develop vaccines that will be effective against dysentery and diarrhea caused by shigella dysentariae . With the growing recognition of the problems associated with sustained vaccine acceptance and usage, researchers and policy makers recognize the importance of conducting theory-based qualitative research to inform vaccine development program efforts . Accordingly we undertook this qualitative study involving 81 residents of one of China's rural communities with high rates of dysentery . The semi-structured interviews suggest that a Western model of behavioral change offered a useful research construct . Consistent with the model is the community's strong perception of 'response efficacy' of vaccines, particularly in comparison with water and sanitation and disease treatment . Residents were eager to vaccinate their children despite variable perception of disease severity, while they were less consistent in their interest in vaccinating adults; this enthusiasm for vaccinating children was attributed to China's 'one child per couple' policy . Intervention implications are discussed.

Bioinformatics, 2004 Dec 12, 20(18), 3455 - 61 Epub 2004 Jul 22.
A probabilistic measure for alignment-free sequence comparison; Pham TD et al.; MOTIVATION: Alignment-free sequence comparison methods are still in the early stages of development compared to those of alignment-based sequence analysis . In this paper, we introduce a probabilistic measure of similarity between two biological sequences without alignment . The method is based on the concept of comparing the similarity/dissimilarity between two constructed Markov models . RESULTS: The method was tested against six DNA sequences, which are the thrA, thrB and thrC genes of the threonine operons from Escherichia coli K-12 and from Shigella flexneri; and one random sequence having the same base composition as thrA from E.coli . These results were compared with those obtained from CLUSTAL W algorithm (alignment-based) and the chaos game representation (alignment-free) . The method was further tested against a more complex set of 40 DNA sequences and compared with other existing sequence similarity measures (alignment-free) . AVAILABILITY: All datasets and computer codes written in MATLAB are available upon request from the first author.

J Neuroimmunol, 2004 Aug, 153(1-2), 36 - 9
Involvement of the neuropeptide corticotropin-releasing hormone in an animal model of Shigella-related seizures; Yuhas Y et al.; Convulsions are common complications of shigellosis in children . Corticotropin-releasing hormone (CRH), a stress neurohormone, has been implicated in the susceptibility of young children to seizures . We investigated the role of CRH in Shigella-related seizures . Pretreatment with Shigella dysenteriae sonicate enhanced mice response to pentylenetetrazole (PTZ)-induced seizures . Preinjection of antalarmin, a CRH-receptor 1 antagonist, decreased both the mean convulsion score (MCS: 1.6 vs . 1.1, p<0.05) and the percent of mice having seizures (48% vs . 28%, p=0.03) . This indicates that CRH plays a role in the increased susceptibility to seizures following exposure to S . dysenteriae.

J Cell Biol, 2004 Jul 19, 166(2), 225 - 35
Cortactin and Crk cooperate to trigger actin polymerization during Shigella invasion of epithelial cells; Bougneres L et al.; Shigella, the causative agent of bacillary dysentery, invades epithelial cells in a process involving Src tyrosine kinase signaling . Cortactin, a ubiquitous actin-binding protein present in structures of dynamic actin assembly, is the major protein tyrosine phosphorylated during Shigella invasion . Here, we report that RNA interference silencing of cortactin expression, as does Src inhibition in cells expressing kinase-inactive Src, interferes with actin polymerization required for the formation of cellular extensions engulfing the bacteria . Shigella invasion induced the recruitment of cortactin at plasma membranes in a tyrosine phosphorylation-dependent manner . Overexpression of wild-type forms of cortactin or the adaptor protein Crk favored Shigella uptake, and Arp2/3 binding-deficient cortactin derivatives or an Src homology 2 domain Crk mutant interfered with bacterial-induced actin foci formation . Crk was shown to directly interact with tyrosine-phosphorylated cortactin and to condition cortactin-dependent actin polymerization required for Shigella uptake . These results point at a major role for a Crk-cortactin complex in actin polymerization downstream of tyrosine kinase signaling . Copyright The Rockerfeller University Press

Anal Chem, 2004 Jul 15, 76(14), 4093 - 7
Detection of approximately 10(3) copies of DNA by an electrochemical enzyme-amplified sandwich assay with ambient O(2) as the substrate; Zhang Y et al.; The electrochemical sandwich-type, enzyme-amplified assay of Zhang, Kim, and Heller (Anal . Chem . 2003, 75, 3267-3269) was simplified by replacing the amplifying horseradish peroxidase with bilirubin oxidase (BOD) . BOD catalyzes the reduction of ambient O(2) to water and obviates the need for adding H(2)O(2) . Femtomolar (10(-)(15) M) concentrations of DNA were detected at a 10-microm-diameter tip of a carbon fiber electrode . Correspondingly, a few thousand copies of DNA were detected in approximately 5-microL samples . The sandwich is formed in an electron-conducting redox hydrogel, to the polymer of which a DNA capture sequence is bound . Capture of the analyte DNA and its hybridization with a BOD-labeled complementary DNA sequence, electrically connects the BOD label to the electron-conducting redox polymer, which is in electrical contact with the electrode . Placing the BOD in contact with the redox polymer thus converts the noncatalytic base layer into a catalyst for the electroreduction of O(2) to water at +0.12 V (vs Ag/AgCl) (Figure 1) . In an exemplary assay, approximately 3000 copies of the iron transporting sequence of the sit gene of Shigella flexneri were detected without PCR amplification.

Gut, 2004 Aug, 53(8), 1096 - 101
Bacillary dysentery as a causative factor of irritable bowel syndrome and its pathogenesis; Wang LH et al.; BACKGROUND AND AIMS: The incidence of irritable bowel syndrome (IBS) or functional bowel disorders (FBD) after bacillary dysentery (BD) has not been extensively evaluated, and little is known of the pathogenesis of post-infective (PI) IBS . Therefore, we investigated the incidence of IBS and FBD in a Chinese patient population who had recovered from BD . To further elucidate its pathogenesis, neuroimmunological changes, including interleukins (IL), mast cells, neuropeptides, and the relationship between mast cells and intestinal nerves, were investigated . METHODS: A cohort study of 295 patients who had recovered from BD (shigella identified from stool in 71.4%) and 243 control subjects consisting of patient siblings or spouses who had not been infected with BD were included in the study . All subjects were followed up using questionnaires for 1-2 years to explore the incidence of FBD and IBS, as defined by the Rome II criteria . In 56 cases of IBS (PI and non-PI) from another source, the number of mast cells in biopsy specimens from the intestinal mucosa were stained with antitryptase antibody and counted under light microscopy . Also, the relationship of mast cells to neurone specific enolase (NSE), substance P (SP), 5-hydroxytryptamine (5-HT), or calcitonin gene related peptide positive nerve fibres was observed using double staining with alcian blue and neuropeptide antibodies . In 30 cases of IBS (PI-IBS, n = 15) taken at random from the 56 cases, expression of interleukin (IL)-1alpha, IL-1beta, and IL-1 receptor antagonist (IL-1ra) mRNAs in intestinal mucosa were identified using reverse transcription-polymerase chain reaction . The above results were compared with 12 non-IBS controls . RESULTS: In the BD infected cohort, the incidences of FBD and IBS were 22.4% and 8.1% (in total)-10.2% (among those in who shigella were identified) respectively, which were significantly higher (p<0.01) than the incidences of FBD (7.4%) and IBS (0.8%) in the control cohort . A longer duration of diarrhoea (>or=7 days) was associated with a higher risk of developing FBD (odds ratio 3.49 (95% confidence interval 1.71-7.13)) . Expression of IL-1beta mRNA in terminal ileum and rectosigmoid mucosa was significantly higher in PI-IBS patients (p<0.01) . The number of mast cells in the terminal ileum mucosa in PI-IBS (11.19 (2.83)) and non-PI-IBS patients (10.78 (1.23)) was significantly increased compared with that (6.05 (0.51)) in control subjects (p<0.01) . Also, in the terminal ileum and rectosigmoid mucosa of IBS patients, the density of NSE, SP, and 5-HT positively stained nerve fibres increased (p<0.05) and appeared in clusters, surrounding an increased number of mast cells (p<0.01 compared with controls) . CONCLUSIONS: BD is a causative factor in PI-IBS . The immune and nervous system may both play important roles in the pathogenesis of PI-IBS.

Vaccine, 2004 Jul 29, 22(21-22), 2750 - 6
Identification of novel immunogenic proteins of Shigella flexneri 2a by proteomic methodologies; Peng X et al.; Shigella spp . are one of the most important etiological factors for people who are living in developing countries and travelers to tropical countries . High priority has been given by the World Health Organization to the development of vaccines to control Shigellosis caused by these bacteria . However, information regarding to profile of immunogenic proteins of Shigella is not available now . In the present study, sub-immunoproteomics was applied to screen novel immunogenic proteins which could be reacted with antisera produced by challenge of a whole bacterium . Our results indicated that 13 immunogens were identified, in which seven proteins and six proteins from outer membrane and soluble proteome, respectively . Of the 13 proteins, 12 showed to be novel immunogens . These results suggest that immunoproteomics can greatly improve the chances of identification and result in discovery of novel immunogenic proteins.

Virology, 2004 Aug 1, 325(2), 192 - 9
Mucosal immunization with attenuated Shigella flexneri harboring an influenza hemagglutinin DNA vaccine protects mice against a lethal influenza challenge; Vecino WH et al.; Mucosal surfaces are important for the induction of immunity against influenza virus . In a murine intranasal immunization model, we demonstrated that the attenuated Shigella flexneri Deltaasd strain 15D, carrying a DNA construct encoding the influenza virus hemagglutinin (HA), induces protective immunity against a lethal respiratory challenge with influenza A/WSN/33 . Influenza virus-specific IFN-gamma T cells were detected among splenocytes, and anti-HA IgG was detected in serum post-immunization, albeit at low levels . Following influenza virus challenge, an accelerated anti-HA IgA antibody response was detected in bronchoalveolar lavage (BAL) washings from mice vaccinated with attenuated shigella containing the HA construct . These results suggest that S . flexneri Deltaasd strain 15D is a promising vector for mucosal DNA vaccine immunization against influenza virus and other mucosal pathogens.

Biometals, 2004 Jun, 17(3), 261 - 5
Effect on bovine lactoferrin on the activation of the enteroinvasive bacterial type III secretion system; Santapaola D et al.; Shigella and enteroinvasive Escherichia coli (EIEC) strains secrete virulence proteins by a complex machinery called the type III secretion (TTS) apparatus . Secretion of virulence proteins is a tightly-regulated phenomenon such that the TTS system is weakly active when bacteria are grown in common laboratory media . Activation of the TTS system is triggered by contact with eukaryotic cells, or can be artificially stimulated by the addition of Congo red dye to the growth medium . Exploiting the ability of bovine lactoferrin (bLf) to bind iron we have found that the TTS of EIEC strain HN280 seems to be activated in conditions of low-iron availability, obtained by incubation of bacteria with bLf enclosed within a dialysis bag . Activation of secretion was assessed by measuring the release of IpaB and C, chosen as reporters of secreted virulence proteins . The contribution of small bLf-derived components, diffusing across the dialysis membrane, in the release of Ipa proteins has also been determined . Activation of secretion was not due to bLf-induced damage of the HN280 outer membrane and was not associated with increased transcription of the mxi operon . Thus, low-iron availability might be an environmental signal perceived by enteroinvasive micro-organisms in order to modulate secretion of virulence proteins.

Biometals, 2004 Jun, 17(3), 257 - 60
Lactoferrin disruption of bacterial type III secretion systems; Ochoa TJ et al.; Many gram-negative bacteria share a closely related mechanism for secretion of virulence proteins . This complex machine, the type III secretion system, secretes virulence proteins in response to sensing the presence of target mammalian cells . We have found that recombinant human lactoferrin impairs the function of this system in two model organisms: Shigella and Enteropathogenic E . coli (EPEC) . In the case of Shigella, there is loss and degradation of two proteins secreted by the type III mechanism, invasion plasmid antigens B and C (IpaB and IpaC); these proteins normally form a complex that causes Shigella to be taken up by host mammalian cells . In the case of EPEC, lactoferrin causes loss and degradation of E . coli secreted proteins A, B and D (EspABD) particularly EspB . These proteins are components of type III machinery and are known to be key elements of EPEC pathogenesis . Studies using purified EspB demonstrated that lactoferrin has a direct proteolytic effect on EspB that can be prevented by serine protease inhibitors . A synthetic peptide of the N-terminal 33 amino acids of lactoferrin caused loss of cell associated EspB but, unlike the whole lactoferrin molecule, did not caused degradation of EspB . Thus, in both model systems, brief exposure to lactoferrin causes loss and degradation of type III secretion system virulence proteins.

Przegl Epidemiol, 2004, 58(1), 77 - 84
{Dysentery in Poland in 2002}; Stypulkowska-Misiurewicz H et al.; The notified number of dysentery cases was still very low, but a little higher than in 2001 . Only 220 cases were notified in the year 2002 (incidence 0.58/100,000 population) while 128 cases were notified in 2001 (incidence 0.33/100,000) . No one death case was notified . Four outbreaks of dysentery were observed: among them one foodborne infection due to S . sonnei involving 109 participants of a summer holiday camp for children from the poor families and one due to S . flexneri 2a involving 16 patients of psychiatric hospital were registered . S . sonnei remains the dominant etiological agent was isolated in 92% of notified dysentery cases and S . flexneri was isolated only in 8% of cases . S . dysenteriae and S . boydii were not found among persons examined bacteriologically by laboratory service of Epidemiological and Sanitary Service . Two tests of the external control of laboratory diagnosis were performed: one concerning the quality of the selective media used in the Sanitary-Epidemiological Stations (SSE) in Poland for routine bacteriological diagnosis of humans intestinal infections . It was shown that the specimens of the SS medium were acceptable only for diagnosis of S . enteritidis infections . In nearly 1/3 of SSE bacteriological laboratories was inefficient for diagnosis of S . flexneri infections and in some laboratories even not for diagnosis of infections due to S . typhi and S . sonnei . In another test the control strain of S . boydii 6 was send to SSE laboratories . The strain was properly diagnosed by all but two laboratories . Besides it was shown that the control strain was not able to grow on selective bacteriological media used for routine work . So there is a need to change the procedure used in those laboratories . In the period of low frequency of Shigella infections the external control of the quality of bacteriological media and laboratory procedures is needed and should be done regularly.

J Infect Dis, 2004 Jul 15, 190(2), 330 - 4 Epub 2004 Jun 09.
Variation in the Shiga toxin region of 20th-century epidemic and endemic Shigella dysenteriae 1 strains; Greco KM et al.; The Shiga toxin (Stx) region of Shigella dysenteriae 1 lies on a defective prophage homologous to lambdoid bacteriophages in Stx-producing Escherichia coli . S . dysenteriae 1 strains obtained in locations throughout the world over the course of the past 60 years were assessed for variations in the Stx region by use of polymerase chain reaction and sequence analysis . The defective prophage was present in all strains examined, suggesting that all S . dysenteriae 1 isolates derive from a clone that resulted from a single phage-integration event . All western-hemisphere strains have an additional iso-IS1 insertion element upstream of stxAB, implying that there has been minimal exchange of strains between hemispheres in recent decades.

J Antimicrob Chemother, 2004 Aug, 54(2), 418 - 23 Epub 2004 Jun 23.
Effect of inoculum density on susceptibility of Plesiomonas shigelloides to cephalosporins; Wiegand I et al.; OBJECTIVES: Resistance of Plesiomonas shigelloides to cephalosporins at higher cell densities has been reported . We investigated whether these inoculum effects are due to the production of beta-lactamases . METHODS: beta-Lactamase production of five P . shigelloides strains was characterized by activity tests, SDS-PAGE and isoelectric focusing . For all strains, MIC values of different cephalosporins were determined by microdilution methodology using inocula of 1 x 10(5) cfu/mL and 1 x 10(6) cfu/mL . Subsequently, the morphology of cells was determined by light microscopy . For one isolate, kill kinetics of cefpodoxime were determined using batch cultures with the lower and higher inocula . RESULTS: Four of five P . shigelloides strains were shown to be beta-lactamase-positive, producing different amounts of constitutively expressed non-inducible enzymes . Inoculum effects for cephalosporin susceptibility were observed for all strains . Examination of cells revealed a very strong filamentation, with filament sizes ranging from 100 microm up to 2 mm . The kill kinetics with cefpodoxime showed similar killing capacities of the antibiotic at both inoculum sizes . CONCLUSIONS: The reported resistance of P . shigelloides to cephalosporins at higher cell densities is not due to an inoculum-dependent regulation of beta-lactamases, but can be explained by the formation of extensive filaments.

Am J Trop Med Hyg, 2004 Jun, 70(6), 663 - 5
Short report: High incidence of shigellosis among Peruvian soldiers deployed in the Amazon River basin; Jones FR et al.; We investigated the etiology of acute diarrhea among Peruvian military recruits undergoing three months of basic combat training near the Amazonian city of Iquitos . From January through September 2002, 307 of 967 recruits were seen at the Health Post for diarrhea (attack rate {AR} = 31.8%, incidence = 1.28 95% confidence interval {CI} = 1.14-1.43} episodes/person-year) . Shigella spp . were the most common bacterial pathogen recovered from recruits experiencing diarrhea episodes . These bacteria were isolated from 89 (40%) of 225 diarrheal stools examined (AR = 7.6%, incidence = 0.30 {95% CI = 0.24-0.38} episodes/person-year) . Most (83 of 90; 92%) of the Shigella isolates were S . flexneri, of which 57 (69%) were serotype 2a . Seventy-six percent of Shigella isolates were resistant to sulfamethoxazole/trimethoprim and all were sensitive to ciprofloxacin . Peruvian soldiers may be an excellent population in which to test the efficacy of S . flexneri vaccines in advanced development.

Emerg Infect Dis, 2004 Jun, 10(6), 1147 - 9
Multistate shigellosis outbreak and commercially prepared food, United States; Kimura AC et al.; In 2000, shigellosis traced to a commercially prepared dip developed in 406 persons nationwide . An ill employee may have inadvertently contaminated processing equipment . This outbreak demonstrates the vulnerability of the food supply and how infectious organisms can rapidly disseminate through point-source contamination of a widely distributed food item.

Zhonghua Liu Xing Bing Xue Za Zhi, 2004 Mar, 25(3), 245 - 7
{Study on the molecular mechanism of quinolone resistance in Shigellae spp}; Zhu JY et al.; OBJECTIVE: To study the resistance and its mechanism of Shigellae spp . to quinolones . METHODS: Seventy-three clinical isolates were collected . Susceptibility tests of pipemidic adcid (PI), ofloxacin (OFL), norfloxacin (NOR), and ciprofloxacin (CIP) were performed in all clinical isolates and Shigella 51573 . The N-terminal coding region of gyrA and parC were amplified by polymerase chain reaction (PCR) respectively . Restriction fragment length polymorphism (RFLP) was applied to all PCR procucts of gyrA and parC, and single strand conformational polymorphism analysis (SSCP) was also applied to PCR procucts of parC . RESULTS: The resistance rates for all the Shigella spp . to PI, CIP, NOR and OFL were 79.5%, 60.3%, 41.1% and 36.9% . Sixty-seven strains (91.8%) were quinolone-reduced-sensitive isolates, in which 61 strains (91%) were found carrying mutations in gyrA with 5 strains (7.5%) found carrying mutations in parC . No mutation was found in 6 quinolone-sensitive isolates or Shigella 51573 . CONCLUSION: The Shigella spp . had high resistance rates to quinolones . The target gene mutations which were mainly found in gyrA and secondarily in parC, played an important role in the quinolone-resistance in Shigella spp.

Rev Panam Salud Publica, 2004 Apr, 15(4), 219 - 24
{Antibiotic resistance and molecular epidemiology of Shigella spp . in northeastern Argentina}; Merino LA et al.; OBJECTIVES: To evaluate the antibiotic resistance of strains of Shigella spp . isolated from feces samples from northeastern Argentina and to characterize the strains in terms of their molecular epidemiology . METHODS: We studied 132 isolates of Shigella spp . obtained from feces samples from 132 patients with diarrhea who were seen at various private and public laboratories in the Argentine provinces of Chaco and Corrientes during the period of 1998 to 2002 . Each strain was characterized according to its serotype, its resistance to 13 individual or combination antibiotics, and its sensitivity to pyocins . With 52 strains selected in relation to their antimicrobial susceptibility profiles we conducted plasmid profile analysis using alkaline lysis, and the repetitive extragenic palindromic sequences were determined by amplifying repetitive DNA segments using polymerase chain reaction . The chi-square test was used to compare proportions, with a level of statistical significance of 0.05 . RESULTS: Shigella flexneri was the most common species (78%), followed by S . sonnei (22%) . In general, the resistance of S . flexneri to the antibiotics studied was greater than that of S . sonnei, and this difference was statistically significant (P < 0.001) for ampicillin, tetracycline, chloramphenicol, and the combination of ampicillin and sulbactam . The S . flexneri strains also showed multiple resistance more often than S . sonnei strains (84.5% vs . 31.0%; P < 0.001) . The strains isolated from S . flexneri were grouped into five pyocin types, three plasmid profiles, and five patterns of repetitive palindromic sequences . The strains of S . sonnei formed three pyocin types, two plasmid profiles, and three patterns of repetitive palindromic sequences . CONCLUSIONS: Given that the Shigella species that were studied showed a high level of resistance to the most frequently used antibiotics, surveillance activities should be implemented in order to detect and control the appearance of new resistant strains . Applying epidemiological typing techniques can provide more precise information about the distribution and evolution of resistant strains of circulating microorganisms.

Clin Microbiol Infect, 2004 Jun, 10(6), 574 - 6
Prevalence of the sat, set and sen genes among diverse serotypes of Shigella flexneri strains isolated from patients with acute diarrhoea; Niyogi SK et al.; New virulence factors associated with the genus Shigella have been described recently . These comprise enterotoxins ShET-1 and ShET-2, encoded by the set and sen genes, respectively, and the secreted autotransporter toxin Sat, encoded by the sat gene . The present study aimed to determine the prevalence of genes encoding these toxins among a collection of Shigella flexneri clinical isolates belonging to different serotypes . While the set gene was detected only in S . flexneri serotypes 2a and 2b, the sen and sat genes were present in diverse serotypes of S . flexneri.

Bioorg Med Chem, 2004 Jul 1, 12(13), 3743 - 54
Synthesis and immunochemical characterization of protein conjugates of carbohydrate and carbohydrate-mimetic peptides as experimental vaccines; Hossany RB et al.; The peptides DRPVPY and MDWNMHAA, which were identified as mimics of the cell-surface polysaccharides of Streptococcus Group A and Shigella flexneri Y, respectively, were used in this study to develop experimental vaccines directed against these two bacteria . Both oligopeptides were synthesized employing the Fmoc solid-phase strategy and linked via the amino end to a bifunctional linker, diethylsquarate . These adducts were then conjugated to the two carrier proteins, bovine serum albumin (BSA) and tetanus toxoid (TT) to yield the peptide conjugate vaccines . The average level of incorporation of DRPVPY and MDWNMHAA on TT was 65% and 75%, respectively, whereas that of both peptide haptens on BSA was 100% . A polysaccharide conjugate against S . flexneri Y, which comprises about 10 tetrasaccharide repeating units, was also prepared based on reductive amination at the reducing end with 1,3-diaminopropane, followed by coupling of the aminated polysaccharide to diethylsquarate, and subsequent coupling of the adduct to TT . An average incorporation of 73% of polysaccharide haptens was achieved . The glycoconjugate and the oligopeptide conjugates were shown to bind effectively to the respective monoclonal antibodies directed against the cell-surface polysaccharides.

J Appl Microbiol, 2004, 97(1), 181 - 90
Identification of Escherichia coli O172 O-antigen gene cluster and development of a serogroup-specific PCR assay; Guo H et al.; AIM: To characterize the locus for O-antigen biosynthesis from Escherichia coli O172 type strain and to develop a rapid, specific and sensitive PCR-based method for identification and detection of E . coli O172 . METHODS AND RESULTS: DNA of O-antigen gene cluster of E . coli O172 was amplified by long-range PCR method using primers based on housekeeping genes galF and gnd Shot gun bank was constructed and high quality sequencing was performed . The putative genes for synthesis of UDP-FucNAc, O-unit flippase, O-antigen polymerase and glycosyltransferases were assigned by the homology search . The evolutionary relationship between O-antigen gene clusters of E . coli O172 and E . coli O26 is shown by sequence comparison . Genes specific to E . coli O172 strains were identified by PCR assays using primers based on genes for O-unit flippase, O-antigen polymerase and glycosyltransferases . The specificity of PCR assays was tested using all E . coli and Shigella O-antigen type strains, as well as 24 clinical E . coli isolates . The sensitivity of PCR assays was determined, and the detection limits were 1 pg microl(-1) chromosomal DNA, 0.2 CFU g(-1) pork and 0.2 CFU ml(-1) water . The total time required from beginning to end of the procedure was within 16 h . CONCLUSION: The O-antigen gene cluster of E . coli O172 was identified and PCR assays based on O-antigen specific genes showed high specificity and sensitivity . SIGNIFICANCE AND IMPACT OF THE STUDY: An O-antigen gene cluster was identified by sequencing . The specific genes were determined for E . coli O172 . The sensitivity of O-antigen specific PCR assay was tested . Although Shiga toxin-producing O172 strains were not yet isolated from clinical specimens, they may emerge as pathogens.

FEMS Microbiol Lett, 2004 Jun 15, 235(2), 259 - 64
Molecular typing and phylogenetic analysis of enteroinvasive Escherichia coli using the fliC gene sequence; Amhaz JM et al.; Non-motile enteroinvasive Escherichia coli (EIEC) is serotyped based only on O antigen polymorphism, since H antigen epitopes, present on the flagellins, cannot be characterised in these bacteria . In this study, we demonstrate the presence of the flagellin-coding fliC gene in non-motile EIEC strains . Moreover, we were able to group the 11 most common non-motile EIEC serotypes into six different RFLP patterns of the fliC gene . Amplicons representing each RFLP pattern were sequenced . Sequencing data were used to construct a phylogenetic tree which showed two main clusters: one sharing similarity with Shigella dysenteriae and pathogenic E . coli, and the other being closer to non-pathogenic E . coli.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Nov, 19(6), 589 - 91
{The observation of the lymphocyte phenotype change in different lymphoid tissues of mice after intranasal immunization with bivalent Shigella vaccines}; Shu CL et al.; AIM: To observe the changes of lymphocyte phenotype in different lymphoid tissues of mice at various time after intranasal immunization with bivalent Shigella vaccines . METHODS: BALB/c mice were randomly divided into three groups, 30 mice per group . Mice were intranasally immunized respectively with PBS, FSM-2117 or FS-5416 four times (bacterial number was sequentially 5x10(6), 1x10(7),4x10(7)and 4x10(7)CFU/mouse) with two week intervals . Lymphocytes in nasal associated lymphoid tissue (NALT), nasal passage(NP), spleen or Peyer's patch (PP) were isolated on the 7th,30th and 90th day after the last immunization . The phenotype of the lymphocytes was detected by FACS . RESULTS: CD3(+) T cells in NALT, NP and PP increased significantly on the 7th day after the immunization, in which most were CD4(+)T cells . B220(+) cells and CD3(+) T cells increased notably in spleens of FSM-2117 group and FS-5416 group, respectively . The same phenotypic changes still maintained in the NALT, NP and spleen on the 30th day after immunization, but only present in NALT and NP on the 90th day . CONCLUSION: Intranasal inoculation with the two bivalent Shigella vaccines can effectively induce immune responses in different mucosal sites and systematically which can durate for a long time and start to weaken from the tissues distal from nose to those proximal.

J Bacteriol, 2004 Jun, 186(12), 3911 - 21
Extensive genomic diversity in pathogenic Escherichia coli and Shigella Strains revealed by comparative genomic hybridization microarray; Fukiya S et al.; Escherichia coli, including the closely related genus Shigella, is a highly diverse species in terms of genome structure . Comparative genomic hybridization (CGH) microarray analysis was used to compare the gene content of E . coli K-12 with the gene contents of pathogenic strains . Missing genes in a pathogen were detected on a microarray slide spotted with 4,071 open reading frames (ORFs) of W3110, a commonly used wild-type K-12 strain . For 22 strains subjected to the CGH microarray analyses 1,424 ORFs were found to be absent in at least one strain . The common backbone of the E . coli genome was estimated to contain about 2,800 ORFs . The mosaic distribution of absent regions indicated that the genomes of pathogenic strains were highly diversified because of insertions and deletions . Prophages, cell envelope genes, transporter genes, and regulator genes in the K-12 genome often were not present in pathogens . The gene contents of the strains tested were recognized as a matrix for a neighbor-joining analysis . The phylogenic tree obtained was consistent with the results of previous studies . However, unique relationships between enteroinvasive strains and Shigella, uropathogenic, and some enteropathogenic strains were suggested by the results of this study . The data demonstrated that the CGH microarray technique is useful not only for genomic comparisons but also for phylogenic analysis of E . coli at the strain level.

Mol Microbiol, 2004 Jun, 52(5), 1329 - 36
Regulated site-specific recombination of the she pathogenicity island of Shigella flexneri; Sakellaris H et al.; The she pathogenicity island (PAI) is a chromosomal, laterally acquired, integrative element of Shigella flexneri that carries genes with established or putative roles in virulence . We demonstrate that spontaneous, precise excision of the element from its integration site in the 3' terminus of the pheV tRNA gene is mediated by an integrase gene (int) and a gene designated rox (regulator of excision), both of which are carried on the she PAI . Integrase-mediated excision occurs via recombination between a 22 bp sequence at the 3' terminus of pheV and an imperfect direct repeat at the pheV-distal boundary of the PAI . Excision leads to the formation of a circular episomal form of the PAI, reminiscent of circular excision intermediates of other mobile elements that are substrates for lateral transfer processes such as conjugation, packaging into phage particles and recombinase-mediated integration into the chromosome . The circle junction consists of the pheV-proximal and pheV-distal boundaries of the PAI converging on a sequence identical to 22 bp at the 3' terminus of pheV . The isolated circle was transferred to Escherichia coli where it integrated specifically into phe tRNA genes, as it does in S . flexneri, independently of recA . We also demonstrate that Rox stimulates, but is not essential for, excision of the she PAI in an integrase-dependent manner . However, Rox does not stimulate excision by activating the transcription of the she PAI integrase gene, suggesting that it has an excisionase function similar to that of a related protein from the P4 satellite element of phage P2.

World J Gastroenterol, 2004 Jun 1, 10(11), 1594 - 9
Modulation of human enteric epithelial barrier and ion transport function by Peyer's patch lymphocytes; Chen J et al.; AIM: To investigate the role of Peyer's patch lymphocytes in the regulation of enteric epithelial barrier and ion transport function in homeostasis and host defense . METHODS: Mouse Peyer's patch lymphocytes were co-cultured with human intestinal epithelial cell line Caco-2 either in the mixed or separated (isolated but permeable compartments) culture configuration . Barrier and transport functions of the Caco-2 epithelial monolayers were measured with short-circuit current (Isc) technique . Release of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and cytokine mRNA expression was analyzed by semi-quantitative RT-PCR . Barrier and iontransport functions of both culture conditions following exposure to Shigella lipopolysaccharide (LPS) were also examined . RESULTS: The transepithelial resistance (TER) of the epithelial monolayers co-cultured with Peyer's patch lymphocytes was maintained whereas that of the Caco-2 monolayers alone significantly decreased after eight days in culture . The forskolin-induced anion secretion, in either absence or presence of LPS, was significantly suppressed in the both co-cultures as compared with the Caco-2 cells alone . Furthermore, only the mixed co-culture condition induced the expression and release of mIL-6 from Peyer's patch lymphocytes, which could be further enhanced by LPS . However, both co-culture conditions suppressed expression and release of epithelial hIL-8 under the unstimulated conditions, while the treatment with LPS stimulated their hIL-8 expression and release . CONCLUSION: Peyer's patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact and cytokine signaling.

Trends Cell Biol, 1996 Mar, 6(3), 109 - 14
Actin-based bacterial motility: towards a definition of the minimal requirements; Lasa I et al.; At the border line between microbiology and cell biology, the spectacular capacity o f some intracellular bacterial pathogens, including Listeria monocytogenes, Shigella flexneri and several Rickettsias, to use actin polymerization as a driving force for intracellular movement, cell-to-cell spreading and dissemination within the infected tissue is being increasingly studied . Now that it is possible to manipulate the bacterial surface proteins involved in this process - ActA o f L . monocytogenes and IcsA of S . flexneri - these bacterial systems are providing experimental models in which to investigate the role o f actin filament dynamics in cell motility.

Clin Infect Dis, 2004 May 15, 38(10), 1372 - 7 Epub 2004 Apr 28.
Laboratory-confirmed shigellosis in the United States, 1989-2002: epidemiologic trends and patterns; Gupta A et al.; During 1989-2002, a total of 208,368 laboratory-confirmed Shigella infections were reported to the Centers for Disease Control and Prevention . Shigella sonnei accounted for 71.7%, Shigella flexneri accounted for 18.4%, Shigella boydii accounted for 1.6%, and Shigella dysenteriae accounted for 0.7% of infections; for 7.6%, no serogroup was reported . National incidence rates ranged from 7.6 cases per 100,000 persons in 1993 to 3.7 cases per 100,000 persons in 1999 . Incidence rates for S . boydii, S . dysenteriae, and S . flexneri decreased over the 14-year period by 81%, 83%, and 64%, respectively; S . sonnei rates only decreased by 8% . The highest rates were reported from western states (10.0 cases per 100,000 persons) and among children 1-4 years of age (20.6 cases per 100,000 persons) . The female-male S . sonnei incidence rate ratio among 20-39-year-old adults decreased from 2.3 during 1989-1999 to 1.4 during 2000-2002 . Approximately 1% of isolates were from extraenteric sources; 0.25% were from blood . S . sonnei remains an important cause of diarrhea in the United States . Prevention efforts that target high-risk groups are needed.

Clin Infect Dis, 2004 Jun 1, 38(11), 1545 - 51 Epub 2004 May 11.
Pathogen-specific risk factors and protective factors for acute diarrheal illness in children aged 12-59 months in São Paulo, Brazil; Sobel J et al.; Diarrheal diseases are a leading cause of childhood morbidity and mortality in Latin America . Most studies have focused on infants but not on older children . We enrolled 505 children (age, 12-59 months) with diarrhea and age-matched controls in a case-control study in Sao Paulo, Brazil . Independent risk factors for diarrhea included another household member with diarrhea (matched odds ratio {mOR}, 8.1; attributable fraction {AF}, 0.17; P<.001) and consumption of homemade juice (mOR, 1.8; AF, 0.10; P=.01); protective factors included boiling of the baby bottle or nipple (mOR, 0.60; AF, 0.19; P=.026), childcare at home (mOR, 0.58; AF, 0.12; P=.004), and piped sewage (mOR, 0.58; AF, 0.05; P=.047) . Hand washing by the caretaker after helping the child defecate protected against Shigella infection (mOR, 0.35; P<.05) . Preparation of rice, beans, or soup in the morning and serving it to children after noon were associated with enterotoxigenic Escherichia coli infection (mOR, 8.0; P<.05) . In these poor households, 28% of cases of diarrhea in 1-4-year-old children was attributable to easily modifiable exposures.

Am J Trop Med Hyg, 2004 May, 70(5), 536 - 9
Etiology of diarrhea in children less than five years of age in Ifakara, Tanzania; Vargas M et al.; A total of 451 stool specimens were collected from children less than five years of age with acute diarrhea from Ifakara, Tanzania and processed to detect bacterial enteropathogens, parasites, and rotaviruses . These specimens were divided into 348 from the dry season and 103 from the rainy season . Overall, diarrheogenic Escherichia coli (35.7%) were the predominant enteropathogens, with enterotoxigenic E . coli, enteroaggregative E . coli, and enteropathogenic E . coli being the most prevalent . Moreover, enteroaggregative E . coli (63% versus 35.5%; P < 0.05), Shigella spp . (24% versus 12%; P < 0.05), and rotavirus (23% versus 4%; P < 0.05) were more prevalent in the dry season than in the rainy season and enterotoxigenic E . coli (51.6% versus 20%; P < 0.05) and Giardia lamblia (14% versus 1%; P < 0.05) were more prevalent in the rainy season.

J Biol Chem, 2004 Jul 23, 279(30), 31237 - 50 Epub 2004 May 20.
Chromosomal and plasmid-encoded enzymes are required for assembly of the R3-type core oligosaccharide in the lipopolysaccharide of Escherichia coli O157:H7; Kaniuk NA et al.; The type R3 core oligosaccharide predominates in the lipopolysaccharides from enterohemorrhagic Escherichia coli isolates including O157:H7 . The R3 core biosynthesis (waa) genetic locus contains two genes, waaD and waaJ, that are predicted to encode glycosyltransferases involved in completion of the outer core . Through determination of the structures of the lipopolysaccharide core in precise mutants and biochemical analyses of enzyme activities, WaaJ was shown to be a UDP-glucose:(galactosyl) lipopolysaccharide alpha-1,2-glucosyltransferase, and WaaD was shown to be a UDP-glucose:(glucosyl)lipopolysaccharide alpha-1,2-glucosyltransferase . The residue added by WaaJ was identified as the ligation site for O polysaccharide, and this was confirmed by determination of the structure of the linkage region in serotype O157 lipopolysaccharide . The initial O157 repeat unit begins with an N-acetylgalactosamine residue in a beta-anomeric configuration, whereas the biological repeat unit for O157 contains alpha-linked N-acetylgalactosamine residues . With the characterization of WaaJ and WaaD, the activities of all of the enzymes encoded by the R3 waa locus are either known or predicted from homology data with a high level of confidence . However, when core oligosaccharide structure is considered, the origin of an additional alpha-1,3-linked N-acetylglucosamine residue in the outer core is unknown . The gene responsible for a nonstoichiometric alpha-1,7-linked N-acetylglucosamine substituent in the heptose (inner core) region was identified on the large virulence plasmids of E . coli O157 and Shigella flexneri serotype 2a . This is the first plasmid-encoded core oligosaccharide biosynthesis enzyme reported in E . coli.

Org Biomol Chem, 2004 May 21, 2(10), 1518 - 27 Epub 2004 Apr 26.
Preparation of synthetic glycoconjugates as potential vaccines against Shigella flexneri serotype 2a disease; Wright K et al.; The synthesis of three neoglycopeptides incorporating carbohydrate haptens, differing in length, covalently linked to a non natural universal T helper peptide is disclosed . They were synthesized according to a blockwise strategy based on the condensation of appropriate di-, tri-, and tetrasaccharide trichloroacetimidate donors onto an azidoethyl 2-acetamido-2-deoxybeta-D-glucopyranoside acceptor . Use of thiol-maleimide coupling chemistry allowed site-selective efficient conjugation.

J Mol Biol, 2004 May 28, 339(2), 379 - 94
The chromosome of Shigella flexneri bacteriophage Sf6: complete nucleotide sequence, genetic mosaicism, and DNA packaging; Casjens S et al.; Shigella flexneri temperate bacteriophage Sf6 is of interest in part because its prophage expresses the oac gene that alters the antigenic properties of the surface O-antigen polysaccharide of its host bacterium . We have determined the complete sequence of its 39,044 bp genome . The sequence shows that Sf6 is a member of the canonical lambdoid phage group, and like other phages of this type has a highly mosaic genome . It has chromosomal regions that encode proteins >80% identical with at least 15 different previously characterized lambdoid phages and prophages, but 43% of the genome, including the virion assembly genes, is homologous to the genome of one phage, HK620 . An analysis of the nucleotide differences between Sf6 and HK620 indicates that even these similar regions are highly mosaic . This mosaicism suggests ways in which the virion structural proteins might interact with each other . The Sf6 early operons are arranged like a typical lambdoid phage, with "boundary sequences" often found between functional modules in the "metabolic" genome domain . By virtue of high degree of similarity in the encoding genes and their DNA target sites, we predict that the integrase, early transcription anti-terminator, CI and Cro repressors, and CII protein of Sf6 have DNA binding specificities very similar to the homologous proteins encoded by phages HK620, lambda, 434 and P22, respectively . The late operon contains two tRNA genes . The Sf6 terminase genes are unusual . Analysis of in vivo initiation of the DNA packaging series showed that the Sf6 apparatus that recognizes DNA for packaging appears to cleave DNA for initiation of packaging series at many sites within a large region of about 1800 bp that includes a possible pac site . This is unlike previously characterized phage packaging mechanisms.

Int J Food Microbiol, 2004 Jun 1, 93(2), 259 - 62
Quantitative contamination and transfer of Escherichia coli from foods by houseflies, Musca domestica L . (Diptera: Muscidae); De Jesus AJ et al.; The housefly, Musca domestica L . (Diptera: Muscidae), is recognized as an important factor in the dissemination of various infectious diseases such as cholera, shigellosis, and salmonellosis . They can also serve as a cross-contamination vector for other foodborne pathogens . However, the potential for bacterial transfer by houseflies has been demonstrated in a qualitative rather than quantitative manner . In this study, the numbers of bacteria a housefly can carry on its body and transfer to a clean surface after exposure to a sugar-milk aqueous solution, steak, and potato salad contaminated with a fluorescent gene Escherichia coli (8 log10 CFU/ml) were determined . In the first series of experiments to quantify bacterial numbers on the flies, about 40-60 flies were transferred into a sterile cage, exposed to the food for 30 min, the flies immobilized and the attached E . coli on each fly enumerated . Detectable E . coli (>1.7 log10 CFU/fly) were found on 43% (29/67), 53% (23/43), and 62% (32/52) of the flies in the cages with sugar/milk, steak, and potato salad, respectively . For the positive flies, the geometric mean carriage (log10 CFU/fly) was 2.93+/-1.24 for sugar-milk, 3.77+/-1.28 for steak, and 2.25+/-0.64 for the potato salad . In the second series of experiments, the transfer of bacteria by individual flies from contaminated food to the inner surface of a sterile jar per each landing was determined . E . coli transferred from the sugar-milk was 3.5+/-0.7 log10 CFU/fly-landing, 3.9+/-0.7 for steak and 2.61+/-1.16 for the potato salad . From the initial contamination levels of bacteria and the number of transferred bacteria, it can be calculated that flies contaminate clean surfaces with approximately 0.1 mg of food per landing .

J Clin Microbiol, 2004 May, 42(5), 2031 - 5
Detection of Shigella by a PCR assay targeting the ipaH gene suggests increased prevalence of shigellosis in Nha Trang, Vietnam; Vu DT et al.; Shigella spp . are exquisitely fastidious gram-negative organisms which frequently escape detection by traditional culture methods . To get a more complete understanding of the disease burden caused by Shigella in Nha Trang, Vietnam, real-time PCR was used to detect Shigella DNA . Randomly selected rectal swab specimens from 60 Shigella culture-positive patients and 500 Shigella culture-negative patients detected by population-based surveillance of patients seeking care for diarrhea were processed by real-time PCR . The target of the primer pair is the invasion plasmid antigen H gene sequence (ipaH), carried by all four Shigella species and enteroinvasive Escherichia coli . Shigella spp . could be isolated from the rectal swabs of 547 of 19,206 (3%) patients with diarrhea . IpaH was detected in 55 of 60 (93%) Shigella culture-positive specimens, whereas it was detected in 87 of 245 (36%) culture-negative patients free of dysentery (P < 0.001) . The number of PCR cycles required to detect a PCR product was highest for culture-negative, nonbloody diarrheal specimens (mean number of cycles to detection, 36.6) and was lowest for children with culture-positive, bloody diarrheal specimens (mean number of cycles, 25.3) (P < 0.001) . The data from real-time PCR amplification indicate that the culture-proven prevalence of Shigella among patients with diarrhea may underestimate the prevalence of Shigella infections . The clinical presentation of shigellosis may be directly related to the bacterial load.

Mol Microbiol, 2004 May, 52(4), 1045 - 54
Branching sites and morphological abnormalities behave as ectopic poles in shape-defective Escherichia coli; Nilsen T et al.; Certain mutants in Escherichia coli lacking multiple penicillin-binding proteins (PBPs) produce misshapen cells containing kinks, bends and branches . These deformed regions exhibit two structural characteristics of normal cell poles: the peptidoglycan is inert to dilution by new synthesis or turnover, and a similarly stable patch of outer membrane caps the sites . To test the premise that these aberrant sites represent biochemically functional but misplaced cell poles, we assessed the intracellular distribution of proteins that localize specifically to bacterial poles . Green fluorescent protein (GFP) hybrids containing polar localization sequences from the Shigella flexneri IcsA protein or from the Vibrio cholerae EpsM protein formed foci at the poles of wild-type E . coli and at the poles and morphological abnormalities in PBP mutants . In addition, secreted wild-type IcsA localized to the outer membrane overlying these aberrant domains . We conclude that the morphologically deformed sites in these mutants represent fully functional poles or pole fragments . The results suggest that prokaryotic morphology is driven, at least in part, by the controlled placement of polar material, and that one or more of the low-molecular-weight PBPs participate in this process . Such mutants may help to unravel how particular proteins are targeted to bacterial poles, thereby creating important biochemical and functional asymmetries.

Clin Infect Dis, 2004 May 1, 38(9), e96 - 9 Epub 2004 Apr 15.
A pediatric cluster of Shigella dysenteriae serotype 1 diarrhea with hemolytic uremic syndrome in 2 families from France; Houdouin V et al.; We report a cluster of pediatric diarrhea due to Shigella dysenteriae serotype 1 involving 11 children in France, including the index case, who had returned from Senegal . Child-to-child transmission was documented by ribotyping . Five children developed hemolytic uremic syndrome (HUS) . On the basis of our findings, the choice of antimicrobial treatment for infections with S . dysenteriae serotype 1 should take into account widespread drug resistance and the risk of HUS.

J Mol Biol, 2004 May 21, 339(1), 161 - 71
Crystal structure of the oxidized form of the periplasmic mercury-binding protein MerP from Ralstonia metallidurans CH34; Serre L et al.; In Ralstonia metallidurans CH34, the gene merP encodes for a periplasmic mercury-binding protein which is capable of binding one mercury atom . The metal-binding site of MerP consists of the highly conserved sequence GMTCXXC found in the family that includes metallochaperones and metal-transporting ATPases . We purified MerP from R.metallidurans CH34 and solved its crystal structure under the oxidized form at 2.0A resolution . Superposition with structures of other metal-binding proteins shows that the global structure of R.metallidurans CH34 oxidized MerP follows the general topology of the whole family . The largest differences are observed with the NMR structure of oxidized Shigella flexneri MerP . Detailed analysis of the metal-binding site suggests a direct role for Y66 in stabilizing the thiolate group of C17 during the mercury-binding reaction . The metal-binding site of oxidized MerP is also similar to the metal-binding sites of oxidized copper chaperone for superoxide dismutase and Atx1, two copper-binding proteins from Saccharomyces cerevisiae . Finally, the packing of the MerP crystals suggests that F38, a well-conserved residue in the MerP family may be important in mercury binding and transfer . We propose a possible mechanism of mercury transfer between two CXXC motifs based on a transient bi-coordinated mercury intermediate.

Best Pract Res Clin Gastroenterol, 2004 Apr, 18(2), 421 - 45
Microbial-gut interactions in health and disease . Progress in enteric vaccine development; Svennerholm AM et al.; Enteric infections resulting in diarrhoea are among the most important causes of morbidity and mortality, particularly in children in developing countries . They are also a common cause of disease among travellers to Africa, Asia and Latin America . Recently, effective, live and inactivated oral and parenteral vaccines against some of the most severe enteric infections-cholera and typhoid fever-have been licensed in several countries . Different candidate vaccines against rotavirus, Shigella and ETEC diarrhoea have also been developed and tested for safety and immunogenicity in developed as well as in developing countries . The protective efficacy of several of these vaccines has also been tested, either in human volunteer challenge studies or in field trials . In this chapter we describe the properties and availability of the recently licensed vaccines and present an update on the diverse efforts being made to achieve new or improved vaccines against the most prevalent enteropathogens.

BMC Microbiol . 2004 Apr 28;4(1):18.
In vitro adhesion and invasion inhibition of Shigella dysenteriae, Shigella flexneri and Shigella sonnei clinical strains by human milk proteins; Willer Eda M et al.; BACKGROUND: Shigella is the etiological agent of shigellosis, a disease responsible for more than 500,000 deaths of children per year, in developing countries . These pathogens colonize the intestinal colon, invade, spreading to the other enterocytes . Breastfeeding plays a very important role in protecting infants from intestinal infections . Amongst milk compounds, glycosylated proteins prevent the adhesion of many enteropathogens in vitro . The aim of this work was to determine the effect of human milk proteins on the colonization potential of Shigella dysenteriae, S . flexneri and S . sonnei . To fulfill this purpose, pooled milk samples from five donors, were fractionated by gel filtration and affinity chromatography . Using tissue culture, the milk fractions obtained were tested in Shigella adhesion and invasion assays . RESULTS: Our revealed showed that both adhesion and invasion of Shigella species were inhibited by low concentration of secretory immunoglobulin A, lactoferrin and free secretory component . This work also showed that, these proteins bind to superficial and whole-cell Shigella proteins . CONCLUSIONS: Our findings suggest that human milk may act inhibiting adhesion and, consequently, invasion of Shigella, thereafter preventing shigellosis in infants.

J Agric Food Chem, 2004 May 5, 52(9), 2485 - 9
Antimicrobial and antioxidant activities of Melissa officinalis L . (Lamiaceae) essential oil; Mimica-Dukic N et al.; The present study describes antimicrobial and free radical scavenging capacity (RSC) together with the effects on lipid peroxidation (LP) of Melissa officinalis essential oil . The chemical profile of essential oil was evaluated by the means of gas chromatography-mass spectrometry (GC-MS) and thin-layer chromatography (TLC) . RSC was assessed measuring the scavenging activity of essential oil on the 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) and OH(*) radicals . The effect on LP was evaluated following the activities on Fe(2+)/ascorbate and Fe(2+)/H(2)O(2) systems of induction . The antimicrobial activity was tested against 13 bacterial strains and six fungi . The examined essential oil exhibited very strong RSC, reducing the DPPH radical formation (IC(50) = 7.58 microg/mL) and OH radical generation (IC(50) = 1.74 microg/mL) in a dose-dependent manner . According to the GC-MS and TLC (dot-blot techniques), the most powerful scavenging compounds were monoterpene aldehydes and ketones (neral/geranial, citronellal, isomenthone, and menthone) and mono- and sesquiterpene hydrocarbons (E-caryophyllene) . Very strong inhibition of LP, particularly in the Fe(2+)/H(2)O(2) system of induction (94.59% for 2.13 microg/mL), was observed in both cases, also in a dose-dependent manner . The most effective antibacterial activity was expressed on a multiresistant strain of Shigella sonei . A significant rate of antifungal activity was exhibited on Trichophyton species.

FEMS Microbiol Lett, 2004 May 1, 234(1), 139 - 47
Comparative analysis of transcriptional regulatory elements of glutamate-dependent acid-resistance systems of Shigella flexneri and Escherichia coli O157:H7; Bhagwat AA et al.; The ability to withstand an acid-challenge of pH 2.5 or less by Shigella flexneri is a necessary trait for virulence and is generally believed to be restricted to the stationary-phase of growth . Earlier reports indicated the glutamate-dependent acid-resistance (GDAR) system of S . flexneri is under the regulation of rpoS, the gene encoding alternative sigma factor that is induced in the stationary-growth phase . The present study reports that unlike Escherichia coli O157:H7, S . flexneri cells when grown in minimal medium, require acid-induction in the stationary-growth phase for a functional GDAR . When grown on complex medium at pH 5.5, GDAR of S . flexneri was vigorous compared to the cells grown at pH 7.5 . No acid-induction was required for the stationary phase E . coli cells grown on either minimal or complex growth media . Distinct differences in the gadA, gadBC, gadE, and hdeA (but not in rpoS) transcript levels were observed in the stationary-growth phase cells between the two pathogens grown on minimal medium . Additionally, rpoS-independent acid-induction of GDAR in the logarithmic growth phase that has been recently observed in E . coli strains {FEMS Microbiol . Lett . 227 (2003) 39-45} was not detected in the S . flexneri rpoS mutant . Although some differences in the DNA sequence at the upstream regulatory elements of gadBC were noticed, they do not appear to be significant and involvement of additional regulators in S . flexneri is anticipated, which also may explain the observed differences in the GDAR of two enteric pathogens.

FEMS Microbiol Lett, 2004 May 1, 234(1), 125 - 32
The O-antigen gene cluster of Shigella boydii O11 and functional identification of its wzy gene; Tao J et al.; Shigella strains are human pathogens and their identification is usually based on their O-antigens . The O-antigen gene cluster of Shigella boydii O11 was sequenced . All the expected genes for the synthesis of the O-antigen were identified on the basis of homology and genes for the biosynthesis of dTDP-l-Rhamnose, genes encoding sugar transferases, as well as genes encoding O unit flippase (wzx) and O-antigen polymerase (wzy) . The identity of the putative wzy gene was confirmed by showing that a wzy deficient mutant strain of S . boydii O11 produced a semi-rough LPS phenotype . The predicted wzx gene has an opposite transcription direction to that of all of the other genes in the S . boydii O11 O-antigen gene cluster . This unusual feature for the wzx gene has only previously been reported in S . boydii O6 . Further comparison revealed an evolutionary relationship between O6 and O11 O-antigen gene clusters . Adjacent-gene PCR showed that Escherichia coli O105 and S . boydii O11, which share the identical O-antigen, also have the same genes and organization for their respective O-antigen gene clusters . Three genes specific for the S . boydii O11 and E . coli O105 gene clusters were identified.

Cell Microbiol, 2004 Jun, 6(6), 535 - 54
Stereotypic and specific elements of the human colonic response to Entamoeba histolytica and Shigella flexneri; Zhang Z et al.; The clinical presentations of bacillary dysentery caused by shigella, and amoebic dysentery caused by the protozoan parasite Entamoeba histolytica, can be indistinguishable, with both organisms causing colonic mucosal damage and ulceration . However, the two organisms are quite distinct, and have very different pathogenic mechanisms . This raises the fundamental question of whether the similar clinical manifestations reflect a stereotypic response of the human gut to mucosal injury, or whether there are differences at the molecular level in the host response to individual gut pathogens . To characterize the human colonic response to each pathogen at the molecular level, we measured the differential transcription of nearly 40,000 human genes in sections of human colonic xenografts obtained 4 and 24 h following infection with Shigella flexneri or E . histolytica . Our results indicate that much of the human colonic response to these two pathogens is stereotypic, with increased expression of genes activated in cells undergoing stress and/or hypoxic responses, genes encoding cytokines, chemokines, and mediators that are involved in immune and inflammatory responses, and genes encoding proteins involved in responses to tissue injury and in tissue repair . The responses to amoeba and Shigella were not identical however, and we found unique elements in each response that may provide new insights into the distinct pathogenic mechanisms of E . histolytica and S . flexneri.

Acta Microbiol Pol, 2003, 52(4), 373 - 8
Synergistic action of photosensitizers and normal human serum in a bactericidal process . I . Effect of chlorophylls; Jankowski A et al.; Susceptibility of some Gram-negative strains against the bactericidal action of normal human serum (NHS) and of chlorophyll, which induces production of reactive oxygen species by light, was studied . A synergistic bactericidal activity of NHS and chlorophyll against E . coli K1 and Shigella flexneri strains was observed.

Clin Infect Dis, 2004 Apr 15, 38 Suppl 3, S175 - 80
Trends in population-based active surveillance for shigellosis and demographic variability in FoodNet sites, 1996-1999; Shiferaw B et al.; Shigella is a common cause of diarrhea in the United States, and accurate surveillance is needed to determine the burden of illness that they cause . Active surveillance for culture-confirmed Shigella infection was done as part of the Foodborne Diseases Active Surveillance Network (FoodNet) . A total of 4317 cases of shigellosis were reported during 1996-1999 in the original FoodNet surveillance areas . The average annual incidence was 7.4 cases/100,000 population . The incidence was similar during 1996-1998, but it declined in 1999 to 5.0 cases/100,000 population . State-to-state variability was seen in the incidence of shigellosis . Higher incidence was observed in California and Georgia . Shigella sonnei accounted for 70% of the infections, followed by Shigella flexneri (24%) . Compared with other age groups, the incidence was highest among children aged 1-4 years of (36.3 cases/100,000 population) . Marked demographic differences were observed between infections with S . sonnei and S . flexneri.

EMBO Rep, 2004 May, 5(5), 477 - 83 Epub 2004 Apr 16.
Structure of Spa15, a type III secretion chaperone from Shigella flexneri with broad specificity; van Eerde A et al.; Type III secretion (TTS) systems are used by many Gram-negative pathogens to inject virulence proteins into the cells of their hosts . Several of these virulence effectors require TTS chaperones that maintain them in a secretion-competent state . Whereas most chaperones bind only one effector, Spa15 from the human pathogen Shigella flexneri and homologous chaperones bind several seemingly unrelated effectors, and were proposed to form a special subgroup . Its 1.8 A crystal structure confirms this specific classification, showing that Spa15 has the same fold as other TTS effector chaperones, but forms a different dimer . The presence of hydrophobic sites on the Spa15 surface suggests that the different Spa15 effectors all possess similar structural elements that can bind these sites . Furthermore, the Spa15 structure reveals larger structural differences between class I chaperones than previously anticipated, which does not support the hypothesis that chaperone-effector complexes are structurally conserved and function as three-dimensional secretion signals.

J Biol Chem, 2004 Jun 25, 279(26), 27511 - 7 Epub 2004 Apr 09.
Structure of shiga toxin type 2 (Stx2) from Escherichia coli O157:H7; Fraser ME et al.; Several serotypes of Escherichia coli produce protein toxins closely related to Shiga toxin (Stx) from Shigella dysenteriae serotype 1 . These Stx-producing E . coli cause outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in humans, with the latter being more likely if the E . coli produce Stx2 than if they only produce Stx1 . To investigate the differences among the Stxs, which are all AB(5) toxins, the crystal structure of Stx2 from E . coli O157:H7 was determined at 1.8-A resolution and compared with the known structure of Stx . Our major finding was that, in contrast to Stx, the active site of the A-subunit of Stx2 is accessible in the holotoxin, and a molecule of formic acid and a water molecule mimic the binding of the adenine base of the substrate . Further, the A-subunit adopts a different orientation with respect to the B-subunits in Stx2 than in Stx, due to interactions between the carboxyl termini of the B-subunits and neighboring regions of the A-subunit . Of the three types of receptor-binding sites in the B-pentamer, one has a different conformation in Stx2 than in Stx, and the carboxyl terminus of the A-subunit binds at another . Any of these structural differences might result in different mechanisms of action of the two toxins and the development of hemolytic uremic syndrome upon exposure to Stx2.

Microbiology, 2004 Apr, 150(Pt 4), 1079 - 84
The role of the Shigella flexneri yihE gene in LPS synthesis and virulence; Edwards-Jones B et al.; Previously, the authors have shown that inactivation of Shigella flexneri yihE, a gene of unknown function upstream of dsbA, which encodes a periplasmic disulphide catalyst, results in a global change of gene expression . Among the severely down-regulated genes are galETKM, suggesting that the yihE mutant, Sh54, may inefficiently produce the UDP-glucose and UDP-galactose required for LPS synthesis . This paper demonstrates that LPS synthesis in Sh54 is impaired . As a result, Sh54 is unable to polymerize host cell actin, due to aberrant localization of IcsA, or to cause keratoconjunctivitis in guinea pigs . Furthermore, Sh54 is more sensitive to some antimicrobial agents, and exhibits epithelial cytotoxicity characteristic of neither wild-type nor dsbA mutants . Supplying galETK in trans restores LPS synthesis and corrects all the defects . Hence, it is clear that the Shigella yihE gene is important not only in regulating global gene expression, as shown previously, but also in virulence through LPS synthesis via regulating the expression of the galETK operon.

Pediatr Infect Dis J, 2004 Apr, 23(4), 367 - 8
Azithromycin as an alternative to nalidixic acid in the therapy of childhood shigellosis; Miron D et al.; In an outbreak of shigellosis in a kibbutz in Northeastern Israel, oral nalidixic acid failed clinically in 35% and microbiologically in 28.4% of 14 children as compared with no clinical and microbiologic failure in 25 children who were treated with oral azithromycin (P < 0.01 and 0.012, respectively) . Thus azithromycin could be a suitable alternative to nalidixic acid.

Epidemiol Infect, 2004 Apr, 132(2), 303 - 16
Molecular epidemiology of Shigella flexneri in a diarrhoea-endemic area of Lima, Peru; Fernandez-Prada CM et al.; A year-long community-based study of diarrhoeal diseases was conducted in Canto Grande, a periurban community in Lima, Peru . In 109 (34%) houses out of 323 that were visited, at least one individual was detected with shigellosis . The frequency of the 161 shigella isolates obtained was as follows: 117 S . flexneri (73%), 21 S . boydii (13%), 15 S . dysenteriae (9%), and 8 S . sonnei (5%) . Using a non-radioactive ipaH gene probe as a molecular epidemiological tool, a total of 41 S . flexneri strains were shown to be distributed in 25 intra-family comparisons by pairs (icp) . Further subdivision, based on a comparison of the serotype, plasmid profile, antibiotic resistances and ipaH hybridization patterns indicated that Group I, with 11 icp (44%), had strains that were identical . Group II with 8 icp (32%), had strains that were different and Group III with 6 icp (24%), had strains with the same serotype and identical ipaH profiles but with differences in other markers . This data indicates that a diversity of shigella clones circulated in this community resulting from both clonal spread and horizontal transfer of genetic elements . Furthermore, ipaH profiling of isolates can be used not only to differentiate between closely related shigella strains but also with other parameters, help to understand the dynamics of the generation of new clones of pathogenic bacteria.

J Mol Biol, 2004 Apr 16, 338(1), 55 - 75
Crystallographic study of inhibitors of tRNA-guanine transglycosylase suggests a new structure-based pharmacophore for virtual screening; Brenk R et al.; The enzyme tRNA-guanine transglycosylase (TGT) is involved in the pathogenicity of Shigellae . As the crystal structure of this protein is known, it is a putative target for the structure-based design of inhibitors . Here we report a crystallographic study of several new ligands exhibiting a 2,6-diamino-3H-quinazolin-4-one scaffold, which has been shown recently to be a promising template for TGT-inhibitors . Crystal structure analysis of these complexes has revealed an unexpected movement of the side-chain of Asp102 . A detailed analysis of the water network disrupted by this rotation has lead to the derivation of a new composite pharmacophore . A virtual screening has been performed based on this pharmacophore hypothesis and several new inhibitors of micromolar binding affinity with new skeletons have been discovered.

Crit Rev Immunol, 2003, 23(5-6), 371 - 401
Shigellosis: innate mechanisms of inflammatory destruction of the intestinal epithelium, adaptive immune response, and vaccine development; Phalipon A et al.; Acute infectious colitis remains a major pediatric issue of worldwide impact because it still represents a significant public health burden among the larger group of diarrheal diseases with the highest mortality rate . It is also a relevant model of inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis . Among cases of acute colitis of infectious origin, shigellosis is certainly the one that has benefited the most from a significant research effort . Shigella, the causative agent, is a Gram-negative bacterium that has the capacity to invade, disrupt, and cause inflammatory destruction of the intestinal epithelial barrier . The molecular and cellular bases of this invasive phenotype essentially encompass crossing of the epithelial lining, apoptotic killing of macrophages, entry into epithelial cells, and escape into the cytoplasm, followed by cell-to-cell spread . Intracellular colonization is likely to protect the micro-organisms from killing by humoral and cellular effectors of the innate immune response . Concurrently, the capacity of Shigella to reprogram invaded epithelial cells to produce proinflammatory mediators plays a major role in the strong inflammatory profile of the disease . This profile is likely to impact on the nature and quality of the adaptive response, which is dominated by humoral protection at the mucosal level.

J Biol Chem, 2004 May 21, 279(21), 22469 - 76 Epub 2004 Mar 17.
Topological analysis of glucosyltransferase GtrV of Shigella flexneri by a dual reporter system and identification of a unique reentrant loop; Korres H et al.; Lipopolysaccharide, particularly the O-antigen component, is one of many virulence determinants necessary for Shigella flexneri pathogenesis . O-Antigen modification is mediated by glucosyltransferase genes (gtr) encoded by temperate serotype-converting bacteriophages . The gtrV gene encodes the GtrV glucosyltransferase, an integral membrane protein that catalyzes the transfer of a glucosyl residue via an alpha1,3 linkage to rhamnose II of the O-antigen unit . This mediates conversion of S . flexneri serotype Y to serotype 5a . Analysis of the GtrV amino acid sequence using computer prediction programs indicated that GtrV had 9-11 transmembrane segments . The computer prediction models were tested by genetically fusing C-terminal deletions of GtrV to a dual reporter system composed of alkaline phosphatase and beta-galactosidase . Sandwiched GtrV-PhoA/LacZ fusions were also constructed at predetermined positions . The enzyme activities of cells with the GtrV-PhoA/LacZ fusions and the particular location of the fusions in the gtrV indicated that GtrV has nine transmembrane segments and one large N-terminal periplasmic loop with the N and C termini located on the cytoplasmic and periplasmic sides of the membrane, respectively . The existence of a unique reentrant loop was discovered after transmembrane segment IV, a feature not documented in other bacterial glycosyltransferases . Its potential role in mediating serotype conversion in S . flexneri is discussed.

Microbes Infect, 2004 Mar, 6(3), 256 - 64
Neuromodulation of experimental Shigella infection reduces damage to the gut mucosa; Svensson L et al.; Bacillary dysentery arises when Shigella invades the colonic and rectal mucosae of the human gut and elicits a strong inflammatory response, which may lead to life-threatening complications . Hence, downregulation of the host inflammatory response is an appealing therapeutical alternative . The gastrointestinal tract is densely innervated, and nerve endings are often found in the vicinity of leukocytes . We have assessed the impact of experimental Shigella infection on levels of neuropeptides in the intestinal mucosa of rabbits . Ligated small intestinal loops were created in rabbits, and either live, pathogenic Shigella flexneri, a nonpathogenic mutant of Shigella, or NaCl was injected into the loops . Infection was allowed to proceed for 8 or 16 h, after which the rabbits were sacrificed and intestinal biopsies collected . Tissue destruction, fluid secretion and degree of bacterial invasion were monitored . Intestinal biopsies were homogenized, and levels of the neuropeptides calcitonin gene-related peptide, substance P, peptide YY (PYY), vasoactive intestinal peptide, somatostatin, galanin, motilin and neurotensin were measured by radioimmunoassay . Loops exposed to invasive Shigella had 5.7 times lower levels of PYY (P = 0.0095) than loops exposed to NaCl, after 16 h of infection . The levels of the other neuropeptides tested were unchanged . Inhibition of nicotinic cholinergic neurotransmission partly protected the intestinal mucosa from destruction elicited by invasive Shigella . These findings indicate that a tissue-invasive bacterium such as Shigella, which is strictly localized to the intestinal mucosa, activates intramural nerve reflexes that presumably involve a nicotinic synapse as well as the neuropeptide PYY.

Diagn Microbiol Infect Dis, 2004 Mar, 48(3), 175 - 9
Molecular characterization of the integrons in Shigella strains isolated from patients with traveler's diarrhea; Navia MM et al.; The prevalence and characterization of Class 1 integrons has been performed in eighty three strains of Shigella spp., isolated between 1995 and 2000 from patients with traveler's diarrhea . A low prevalence (13.25%) was recorded . Nine different integrons were found among 11 multiresistant strains, with a total of 10 different gene cassettes encoding for resistance to trimethoprim (dfrA1, dfrA5, dfrA7, dfrA12, and dfrA15), aminoglycosides (aadA1a and aadA2), beta-lactam antibiotics (oxa2) or ORF with unknown function (orfD and orfF) . A high prevalence of dfr and aad gene cassettes was observed . The low incidence of Class 1 integrons observed in this study is in contrast with the known facility that the Shigella genus has to gain and transfer plasmids.

Mol Biol Evol, 2004 Jun, 21(6), 1085 - 94 Epub 2004 Mar 10.
A specific genetic background is required for acquisition and expression of virulence factors in Escherichia coli; Escobar-Paramo P et al.; In bacteria, the evolution of pathogenicity seems to be the result of the constant arrival of virulence factors (VFs) into the bacterial genome . However, the integration, retention, and/or expression of these factors may be the result of the interaction between the new arriving genes and the bacterial genomic background . To test this hypothesis, a phylogenetic analysis was done on a collection of 98 Escherichia coli/Shigella strains representing the pathogenic and commensal diversity of the species . The distribution of 17 VFs associated to the different E . coli pathovars was superimposed on the phylogenetic tree . Three major types of VFs can be recognized: (1) VFs that arrive and are expressed in different genetic backgrounds (such as VFs associated with the pathovars of mild chronic diarrhea: enteroaggregative, enteropathogenic, and diffusely-adhering E . coli), (2) VFs that arrive in different genetic backgrounds but are preferentially found, associated with a specific pathology, in only one particular background (such as VFs associated with extraintestinal diseases), and (3) VFs that require a particular genetic background for the arrival and expression of their virulence potential (such as VFs associated with pathovars typical of severe acute diarrhea: enterohemorragic, enterotoxigenic, and enteroinvasive E . coli strains) . The possibility of a single arrival of VFs by chance, followed by a vertical transmission, was ruled out by comparing the evolutionary histories of some of these VFs to the strain phylogeny . These evidences suggest that important changes in the genome of E . coli have occurred during the diversification of the species, allowing the virulence factors associated with severe acute diarrhea to arrive in the population . Thus, the E . coli genome seems to be formed by an "ancestral" and a "derived" background, each one responsible for the acquisition and expression of different virulence factors.

Carbohydr Res, 2004 Apr 2, 339(5), 961 - 6
The conformations of the O-specific polysaccharides of Shigella dysenteriae type 4 and Escherichia coli O159 studied with molecular mechanics (MM3) filtered systematic search; Rosen J et al.; The branched O-antigens of Escherichia coli O159 and Shigella dysenteriae type 4 are structurally related and are known to show cross-reactivity with antibodies . In the present study, conformational analyses were performed on these two O-antigens using molecular mechanics MM3(96) with filtered systematic search . The results show very strong steric restrictions for the trisaccharide at the branch point of the E . coli O159 antigen, especially for the beta-d-GlcNAc-(1-->3)-beta-d-GlcNAc linkage of the main chain . For the type 4 O-antigen the calculations show essentially a single conformation with respect to the alpha-d-GlcNAc-(1-->3)-alpha-d-GlcNAc linkage of the main chain and three different favoured conformations for the fucose branch . Consecutive repeating units of the S . dysenteriae type 4 and E . coli O159 O-antigens form linear extended chains with significant flexibility between the branches . Comparative calculations carried out with the SWEET server indicate that our method of filtered systematic search is a superior method in the case of branched, constrained oligosaccharides . Based on the results of the MM3 calculations, we propose that the common epitope explaining the cross-reactivity comprises the fucose branch, the downstream GlcNAc and part of the uronic acid.

Cell Microbiol, 2004 Apr, 6(4), 355 - 66
Shigella interactions with the actin cytoskeleton in the absence of Ena/VASP family proteins; Ally S et al.; Shigella move through the cytosol of infected cells by assembly of a propulsive actin tail at one end of the bacterium . Vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins, is important in cellular actin dynamics and is present on intracellular Shigella . VASP binds both profilin, an actin monomer-binding protein, and vinculin, a component of intercellular contacts that also binds the Shigella actin assembly protein IcsA . It has been postulated that VASP might serve as a linker between vinculin and profilin on intracellular Shigella, thereby delivering profilin to the Shigella actin assembly machinery . We show that Shigella actin-based motility is unaltered in cells that are deficient for the Ena/VASP family of proteins . In these cells, Shigella form normal-appearing actin tails and move at rates that are comparable to the rates of bacterial movement in Ena/VASP-deficient cells complemented with the Ena/VASP family member Mena . Finally, whereas vinculin can bind the Arp2/3 complex, we show that Arp2/3 recruitment to Shigella is not correlated with vinculin recruitment, indicating that the role of vinculin in Shigella motility is not recruitment of Arp2/3 . Thus, although VASP is recruited to the surface of intracellular Shigella, it is not essential for Shigella actin-based motility.

Am J Clin Nutr, 2004 Mar, 79(3), 444 - 50
Effect of zinc supplementation on immune and inflammatory responses in pediatric patients with shigellosis; Raqib R et al.; BACKGROUND: Several studies showed benefits of long-term zinc supplementation on the incidence, severity, and duration of diarrhea and on the incidence of respiratory infections . Prolonged zinc supplementation also improves cell-mediated immunity in severely malnourished children . OBJECTIVE: We studied the effect of short-term zinc supplementation on intrinsic and specific immune and inflammatory responses in moderately malnourished children with acute shigellosis . DESIGN: A randomized, double-blind, placebo-controlled trial was conducted in Shigella-infected children aged 12-59 mo . Elemental zinc (20 mg) and a multivitamin containing vitamins A and D, thiamine, riboflavin, nicotinamide, and calcium at twice the recommended dietary allowance were given daily for 2 wk to the zinc group (n = 28), whereas the multivitamin alone was given to the control group (n = 28) . Standard antibiotic therapy was given to all patients . RESULTS: Serum zinc concentrations increased in both groups during convalescence; however, zinc supplementation showed a significant effect . The lymphocyte proliferation response in the zinc group increased relative to that in the control group (P = 0.002), but no significant effects were seen on concentrations of cytokines (interleukin 2 and interferon gamma) released from mitogen-stimulated mononuclear cells or on concentrations of cytokines (interleukin 2, interferon gamma, and interleukin 1beta) in feces . Among the antigen {lipopolysaccharide and invasion plasmid-encoded antigen (Ipa)}-specific antibodies, plasma Ipa-specific immunoglobulin G responses at day 30 were significantly higher in the zinc group than in the control group . However, the 2 groups did not differ significantly in the other antigen-specific responses in plasma and stool . CONCLUSION: A 14-d course of zinc supplementation during acute shigellosis increases the lymphocyte proliferation response and the Ipa-specific immunoglobulin G response.

Antimicrob Agents Chemother, 2004 Mar, 48(3), 1028 - 31
Role of attP in integrase-mediated integration of the Shigella resistance locus pathogenicity island of Shigella flexneri; Turner SA et al.; The Shigella resistance locus (SRL) pathogenicity island (PAI) in Shigella spp . mediates resistance to streptomycin, ampicillin, chloramphenicol, and tetracycline . It can be excised from the chromosome via site-specific recombination mediated by the P4-related int gene . Here, we show that SRL PAI attP is capable of RecA-independent, site-specific, int-mediated integration into two bacterial tRNA attB sites.

Epidemiol Infect, 2004 Jan, 132(1), 51 - 6
Variability of Shigella flexneri serotypes in Israel during a period of two years: 2000 and 2001; Vasilev V et al.; During a period of 2 years (2000 and 2001) 996 Shigella flexneri strains from sporadic cases in Israel were sent to the National Shigella Reference Centre (NSRC) by hospital and outpatient clinics . The most common serotypes were 2a, 6 and 1b, accounting for 88.4% of all isolates . They were investigated according to the monthly distribution of the strains, and the age and sex of the patients . The severity of the disease was assessed by a hospital/outpatient distribution (H/Od) of the isolates, based on the location of the sending laboratory . The most affected age groups were 0-11 months and 1-4 years, and the prevalent serotype was 2a, while serotype 6 was dominant in the 5-14 years age group . More cases were registered during the hot season, and there were some serotype-related variations . Overall, 62.1% of the samples were from male patients . Serotype 1b was dominant in the male/female ratio, although it was third in general prevalence . According to the H/Od serotype 2a was more common in hospitalized males and serotype 6 in outpatients, both male and female . These variations, as well as changes in serotype prevalence in the past, underscore the importance of serotype monitoring as part of the public health strategies for reducing the burden of Shigella flexneri infections.

Folia Microbiol (Praha), 2003, 48(5), 659 - 63
Some properties of Plesiomonas shigelloides treated with aminoglycosides; Hostacka A et al.; The effect of aminoglycoside antibiotics (amikacin, gentamicin, netilmicin and tobramycin) at sublethal concentrations (sub-MICs) on some properties of Plesiomonas shigelloides strains was evaluated . All agents decreased the bacterial surface hydrophobicity . Amikacin (1/4 of the MIC) and netilmicin (1/4 and 1/8 of the MIC) changed the hydrophobic character of P . shigelloides surface to a hydrophilic one . Treatment of the strains with aminoglycosides decreased also motility, netilmicin being the most effective . No significant changes were found in lipolytic activity of antibiotic-treated strains . In the majority of cases aminoglycosides increased sensitivity of bacteria to hydrogen peroxide . The tested antibiotics did not induce production of short-chained N-acylhomoserine lactones signal molecules . Aminoglycosides at sub-MICs affected important activities of P . shigelloides potentially associated with their virulence in dependence on strain, antibiotic and concentration.

FEMS Microbiol Rev, 2004 Feb, 28(1), 43 - 58
Shigella flexneri infection: pathogenesis and vaccine development; Jennison AV et al.; Shigella flexneri is a gram-negative bacterium which causes the most communicable of bacterial dysenteries, shigellosis . Shigellosis causes 1.1 million deaths and over 164 million cases each year, with the majority of cases occurring in the children of developing nations . The pathogenesis of S . flexneri is based on the bacteria's ability to invade and replicate within the colonic epithelium, which results in severe inflammation and epithelial destruction . The molecular mechanisms used by S . flexneri to cross the epithelial barrier, evade the host's immune response and enter epithelial cells have been studied extensively in both in vitro and in vivo models . Consequently, numerous virulence factors essential to bacterial invasion, intercellular spread and the induction of inflammation have been identified in S . flexneri . The inflammation produced by the host has been implicated in both the destruction of the colonic epithelium and in controlling and containing the Shigella infection . The host's humoral response to S . flexneri also appears to be important in protecting the host, whilst the role of the cellular immune response remains unclear . The host's immune response to shigellosis is serotype-specific and protective against reinfection by the same serotype, making vaccination a possibility . Since the 1940s vaccines for S . flexneri have been developed with little success, however, the growing understanding of S . flexneri's pathogenesis and the host's immune response is assisting in the generation of more refined vaccine strategies . Current research encompasses a variety of vaccine types, which despite disparity in their efficacy and safety in humans represent promising progress in S . flexneri vaccine development.

New Microbiol, 2004 Jan, 27(1), 37 - 47
Bloody diarrhoea cases caused by Shigella and amoeba in Jordan; Battikhi MN; This study was done to evaluate the occurrence of bloody diarrhoea in Jordan in the period 1988-2000, with particular interest in Shigilla and amoeba cases in relation to the surveillance control system and hygiene policy . The cases were collected from various medical diagnostic laboratories . In addition to the clinical presentation, stool specimens or rectal swabs were collected and sub-cultured on the appropriate media . Shigella spp . and amoeba were identified using standard methods . Anti-microbial susceptibility tests were performed . One-way ANOVA and Least Significant Difference (LSD) were used for statistical analysis . The number of stool specimens tested for bloody diarrhoea was 34,529 in the study period . Positive stool samples were reported in 711 (2.1%) patients . Out of these 55% were males and 45 % were females . The highest number of cases 96 (13.5%) was reported in the year 1999 . The highest incidence rate was (20.6) in the year 1996 and the lowest incidence rate (8.9) was in the year 2000 . The highest number of cases during the period 1998-1992 was among age group 1-4 year . During the year 1993-1996 the highest number of cases was among group 5-14 year . The highest number of cases during the year 1997-2000 was among age group 15-24 year . There was a significant variation between numbers of bloody diarrhoeal cases and age groups (P < 0.001) as well as sex (P = 0.028) . No significant variation (P = 0.06) was observed between number of bloody diarrhoea cases and seasons or months . The number of stool specimens tested for amoebiasis during the study period 1994-2000 was 229,040 . Shigella was isolated from 304(0.13%) cases and amoeba was found in 24,211(10.6%) cases . The lowest incidence rate in bloody diarrhoeal cases in the year 2000 indicates good quality hygienic control . The increased resistance of Shigella to antibiotics may produce a future problem, which might require a further prescribing policy for treatment.

J Org Chem, 2004 Feb 20, 69(4), 1060 - 74
Blockwise approach to fragments of the O-specific polysaccharide of Shigella flexneri serotype 2a: convergent Synthesis of a decasaccharide representative of a dimer of the branched repeating unit; Belot F et al.; The D'A'B'(E')C'DAB(E)C decasaccharide representative of a dimer of a frame-shifted pentasaccharide repeating unit of the O-specific polysaccharide of Shigella flexneri 2a was synthesized as its methyl glycoside by condensing a pentasaccharide donor (D'A'B'(E')C') and a pentasaccharide acceptor (DAB(E)C-OMe) . Several convergent routes to these two building blocks, involving either the AB linkage or the BC linkage as the disconnection site, were evaluated in comparison to the linear strategy . The latter was preferred . It is based on the use of the trichloroacetimidate chemistry . The target branched oligosaccharide was designed to probe the recognition at the molecular level of the natural polysaccharide by protective monoclonal antibodies.

J Clin Microbiol, 2004 Feb, 42(2), 933 - 5
Two cases of continuous ambulatory peritoneal dialysis-associated peritonitis due to Plesiomonas shigelloides; Woo PC et al.; We describe herein the first two cases of Plesiomonas shigelloides continuous ambulatory peritoneal dialysis-related peritonitis . Both patients presented with abdominal pain and turbid dialysis effluent with or without fever . Both recovered after 10 days of intraperitoneal administration of cefazolin and tobramycin . The route of transmission may have been direct contamination of the connection device or bacterial translocation through the gastrointestinal tract.

Mol Microbiol, 2004 Jan, 51(2), 523 - 37
The virF promoter in Shigella: more than just a curved DNA stretch; Prosseda G et al.; In the human enteropathogen Shigella transcription of virF, the primary regulator of the invasion functions, is strictly temperature-dependent and is antagonistically mediated by H-NS and FIS, which bind to specific sites on the virF promoter . Here we report on the relevance of DNA geometry to the thermoregulation of virF and demonstrate that the virF promoter hosts a major DNA bend halfway between two H-NS sites . The bent region has been mutagenized in vitro to mimic temperature-induced changes of DNA curvature . Functional analysis of curvature mutants and of promoter constructs in which the two H-NS sites are phased-out by a half-helix turn reveals that modifying the spatial relationships between these sites severely affects the interaction of H-NS with the virF promoter, as well as its in vivo and in vitro temperature-dependent activity . The role of promoter curvature as thermosensor is also compatible with the present observation that, with increasing temperature, the virF bending centre moves downstream at a rate having its maximum around the transition temperature, abruptly unmasking a binding site for the transcriptional activator FIS.

Nature, 2004 Jan 29, 427(6973), 457 - 61
The RickA protein of Rickettsia conorii activates the Arp2/3 complex; Gouin E et al.; Actin polymerization, the main driving force for cell locomotion, is also used by the bacteria Listeria and Shigella and vaccinia virus for intracellular and intercellular movements . Seminal studies have shown the key function of the Arp2/3 complex in nucleating actin and generating a branched array of actin filaments during membrane extension and pathogen movement . Arp2/3 requires activation by proteins such as the WASP-family proteins or ActA of Listeria . We previously reported that actin tails of Rickettsia conorii, another intracellular bacterium, unlike those of Listeria, Shigella or vaccinia, are made of long unbranched actin filaments apparently devoid of Arp2/3 (ref . 4) . Here we identify a R . conorii surface protein, RickA, that activates Arp2/3 in vitro, although less efficiently than ActA . In infected cells, Arp2/3 is detected on the rickettsial surface but not in actin tails . When expressed in mammalian cells and targeted to the membrane, RickA induces filopodia . Thus RickA-induced actin polymerization, by generating long actin filaments reminiscent of those present in filopodia, has potential as a tool for studying filopodia formation.

MMWR Morb Mortal Wkly Rep, 2004 Jan 30, 53(3), 60 - 3
Day care-related outbreaks of rhamnose-negative Shigella sonnei--six states, June 2001-March 2003; Centers for Disease Control and Prevention (CDC); During June 2001--March 2003, outbreaks of Shigella sonnei infections were reported in Delaware, Maryland, New Jersey, North Carolina, South Carolina, and Virginia . Five- to fortyfold increases in statewide shigellosis rates were observed during this period . These increases were attributed primarily to outbreaks in multiple day care settings that became prolonged and community wide . S . sonnei isolates from these states, as well as from New York City and Philadelphia, were similar genetically by pulsed-field gel electrophoresis (PFGE) . Many of these isolates lacked the capacity to ferment rhamnose, which is unusual for S . sonnei . This report summarizes these outbreaks and describes the laboratory characteristics that link them . The findings underscore the importance of rapid and coordinated public health responses to isolated outbreaks of shigellosis.

Infect Immun, 2004 Feb, 72(2), 923 - 30
Two studies evaluating the safety and immunogenicity of a live, attenuated Shigella flexneri 2a vaccine (SC602) and excretion of vaccine organisms in North American volunteers; Katz DE et al.; We report the first community-based evaluation of Shigella flexneri 2a strain SC602, a live, oral vaccine strain attenuated by deletion of the icsA (virG) plasmid virulence gene, given at 10(4) CFU . The primary objectives of this trial were to determine the safety and immunogenicity of the vaccine and to determine the duration of colonization . Four of 34 volunteers experienced transient fevers, and three reported diarrhea during the first 3 days of the study . Half of the volunteers mounted a positive serum immunoglobulin A (IgA) response to S . flexneri lipopolysaccharide . All but one of the volunteers excreted the vaccine in their stools for 1 to 33 days, and this excretion was often intermittent . Data from the community-based study were supplemented with an inpatient trial in which three volunteers received 10(3) and nine received 10(4) CFU . All volunteers who received 10(3) CFU excreted SC602 and had an IgA antibody-secreting cell response . Two of these had a serum IgA response . Six of the nine volunteers who received 10(4) CFU excreted SC602 . One vaccinee had a transient fever and two met the definition of diarrhea . Six volunteers that received 10(4) CFU had an antibody-secreting cell response, and four had a serum IgA response . SC602 has now been tested at 10(4) CFU in a total of 58 volunteers . The cumulative results of these clinical trials, reported here and previously (Coster et al., Infect . Immun . 67:3437-3443, 1999), have demonstrated that SC602 is a substantially attenuated candidate vaccine that can evoke protection against the most severe symptoms of shigellosis in a stringent human challenge model of disease.

Antimicrob Agents Chemother, 2004 Feb, 48(2), 681 - 4
Clonal multidrug-resistant Shigella dysenteriae type 1 strains associated with epidemic and sporadic dysenteries in eastern India; Pazhani GP et al.; Multidrug-resistant strains of Shigella dysenteriae type 1 were implicated in three outbreaks and sporadic cases of dysentery in eastern India in 2002 and 2003 . After a hiatus of 14 years, this pathogen reemerged with an altered antibiotic resistance pattern . In addition to ampicillin, co-trimoxazole, tetracycline, chloramphenicol, and nalidixic acid, all the recent strains were resistant to norfloxacin, lomefloxacin, pefloxacin, and ofloxacin and showed reduced susceptibility to ciprofloxacin . Pulsed-field gel electrophoresis identified a new clone of S . dysenteriae type 1 that was associated with the recent outbreaks and sporadic cases . Based on the spatial and temporal spread of multidrug-resistant S . dysenteriae type 1, we predict that this clonal type may spread further in this region.

Br J Ophthalmol, 2004 Feb, 88(2), 247 - 50
High levels of IgG class antibodies to recombinant HSP60 kDa of Yersinia enterocolitica in sera of patients with uveitis; Cancino-Diaz JC et al.; AIMS: To determine the levels of IgG class antibodies to recombinant heat shock protein 60 kDa of Yersinia enterocolitica (rHSP60Ye), Klebsiella pneumoniae (rHSP60Kp), Escherichia coli (rHSP60Ec), Shigella flexneri (rHSP60Sf), and Streptococcus pyogenes (rHSP60Sp) in the serum of patients with HLA-B27 associated acute anterior uveitis (HLA-B27 associated AAU), idiopathic acute anterior uveitis (idiopathic AAU), pars planitis, Vogt-Koyanagi-Harada (VKH), and healthy subjects . METHODS: The genes that code for HSP60Ye, HSP60Kp, HSP60Ec, HSP60Sf, and HSP60Sp were cloned by PCR from genomic DNA . The rHSPs were purified by affinity using a Ni-NTA resin . The serum levels of IgG class antibodies to rHSP60s were determined by ELISA in patients with uveitis (n = 42) and in healthy subjects (n = 25) . RESULTS: The majority of patients with uveitis had higher levels of IgG class antibodies to rHSP60Ye compared with levels of healthy subjects (p = 0.01), although these differences were only observed in the HLA-B27 associated AAU (p = 0.005) and in pars planitis patients (p = 0.001) . The levels of IgG antibodies to the rHSP60Kp, rHSP60Sf, rHSP60Ec, and rHSP60Sp were similar in patients with uveitis and in healthy subjects (p>0.05) . CONCLUSION: The results suggest that HSP60Ye could be involved in the aetiology of HLA-B27 associated AAU and pars planitis.

Microbiol Immunol, 2004, 48(1), 49 - 52
A multi-prefectural outbreak of Shigella sonnei infections associated with eating oysters in Japan; Terajima J et al.; Among roughly one thousand incidents of shigellosis annually in Japan, approximately 70% of the cases are estimated to be associated with overseas travel . However, at the end of 2001, reports of domestically acquired Shigella sonnei infections suddenly increased . We report here the first multi-prefectural outbreak of Shigella sonnei infections linked to the consumption of imported oysters in Japan at the end of 2001 . Isolates of S . sonnei from patients epidemiologically linked to eating contaminated oysters and from the imported oysters themselves showed an indistinguishable pulsed-field gel electrophoresis pattern and drug resistance pattern.

BMC Microbiol . 2004 Jan 20;4(1):4.
Analysis of the lambdoid prophage element e14 in the E . coli K-12 genome; Mehta P et al.; BACKGROUND: Many sequenced bacterial genomes harbor phage-like elements or cryptic prophages . These elements have been implicated in pathogenesis, serotype conversion and phage immunity . The e14 element is a defective lambdoid prophage element present at 25 min in the E . coli K-12 genome . This prophage encodes important functional genes such as lit (T4 exclusion), mcrA (modified cytosine restriction activity) and pin (recombinase) . RESULTS: Bioinformatic analysis of the e14 prophage sequence shows the modular nature of the e14 element which shares a large part of its sequence with the Shigella flexneri phage SfV . Based on this similarity, the regulatory region including the repressor and Cro proteins and their binding sites were identified . The protein product of b1149 was found to be a fusion of a replication protein and a terminase . The genes b1143, b1151 and b1152 were identified as putative pseudogenes . A number of duplications of the stfE tail fibre gene of the e14 are seen in plasmid p15B . A protein based comparative approach using the COG database as a starting point helped detect lambdoid prophage like elements in a representative set of completely sequenced genomes . CONCLUSIONS: The e14 element was characterized for the function of its encoded genes, the regulatory regions, replication origin and homology with other phage and bacterial sequences . Comparative analysis at nucleotide and protein levels suggest that a number of important phage related functions are missing in the e14 genome including parts of the early left operon, early right operon and late operon . The loss of these genes is the result of at least three major deletions that have occurred on e14 since its integration . A comparative protein level approach using the COG database can be effectively used to detect defective lambdoid prophage like elements in bacterial genomes.

J Bacteriol, 2004 Feb, 186(3), 699 - 705
Regulation of IcsP, the outer membrane protease of the Shigella actin tail assembly protein IcsA, by virulence plasmid regulators VirF and VirB; Wing HJ et al.; The Shigella outer membrane protease IcsP removes the actin assembly protein IcsA from the bacterial surface, and consequently modulates Shigella actin-based motility and cell-to-cell spread . Here, we demonstrate that IcsP expression is undetectable in mutants lacking either of two transcriptional activators, VirF and VirB . In wild-type Shigella spp., virB expression is entirely dependent on VirF; therefore, to circumvent this regulatory cascade, we independently expressed VirF or VirB in Shigella strains lacking both activators and measured both IcsP levels and transcription from the icsP promoter . Our results show that VirB significantly enhanced icsP transcription, even in the absence of VirF . In contrast, when VirF was induced in the absence of VirB, VirF had variable effects . The regulation of icsP is distinctly different from the regulation of the gene encoding its major substrate, icsA, which is activated by VirF and not VirB . We propose that the different pathways regulating icsA and icsP may be critical to the modulation of IcsA-mediated actin-based motility by IcsP.

J Inorg Biochem, 2004 Feb, 98(2), 387 - 92
A Schiff base complex of chromium(III): an efficient inhibitor for the pathogenic and invasive potential of Shigella dysenteriae; Shrivastava HY et al.; A Schiff base complex of chromium(III), transdiaqua{N,N'ethylenebis (salicylideneimine)chromium(III)}perchlorate, {Cr(salen)(OH(2))(2)}(+), was found to have an inhibitory effect on the growth of Shigella dysenteriae . The chromium(III) complex was found to cure (remove) the invasive plasmid and thereby render the microbe more sensitive to the tested antibiotics . The loss in the catalytic activity of the isolated endo-alpha-N-acetyl galactosaminidase on mucin as a substrate was also observed in the presence of {Cr(salen)(OH(2))(2)}(+) . This suggests that {Cr(salen)(OH(2))(2)}(+) is toxic to the microbe and could make the microbe non-pathogenic and non-invasive, thus establishing its role in microbiological applications to reduce the toxic potentials of a microbe.

Emerg Infect Dis, 2003 Nov, 9(11), 1471 - 4
Shigella dysenteriae serotype 1, Kolkata, India; Dutta S et al.; Since July 2002, bacteriologically confirmed shigellosis cases have increased, and multidrug-resistant Shigella dysenteriae serotype 1 strains have reemerged in patients hospitalized with diarrhea in Kolkata, India . The isolated strains of S . dysenteriae 1 showed resistance to chloramphenicol (80%), ampicillin (100%), tetracycline (100%), co-trimoxazole (100%), nalidixic acid (100%), norfloxacin (100%), and ciprofloxacin (100%) . Emergence of fluoroquinolone resistance in S . dysenteriae 1 strains complicated treatment of shigellosis patients . Six strains belonging to provisional serovars of S . dysenteriae were also identified for the first time in patients hospitalized with diarrhea in Kolkata, India.

J Commun Dis, 2002 Dec, 34(4), 270 - 5
First report of Vibrio cholerae infection from Andaman and Nicobar, India; Shah WA et al.; Out of 34 Stool Samples collected during an outbreak of diarrhoea, Vibrio cholerae 01 was isolated from 10 samples contrary to earlier reports that Shigella species was the only cause of diarrhoeal disease in Andaman & Nicobar Islands.

J Bacteriol, 2004 Jan, 186(2), 383 - 92
Structural and genetic characterization of the Shigella boydii type 13 O antigen; Feng L et al.; Shigella is an important human pathogen . It is generally agreed that Shigella and Escherichia coli constitute a single species; the only exception is Shigella boydii type 13, which is more distantly related to E . coli and other Shigella forms and seems to represent another species . This gives S . boydii type 13 an important status in evolution . O antigen is the polysaccharide part of the lipopolysaccharide in the outer membrane of gram-negative bacteria and plays an important role in pathogenicity . The chemical structure and genetic organization of the S . boydii type 13 O antigen were investigated . The O polysaccharide was found to be acid labile owing to the presence of a glycosyl phosphate linkage in the main chain . The structure of the linear pentasaccharide phosphate repeating unit (O unit) was established by nuclear magnetic resonance spectroscopy, including two-dimensional COSY, TOCSY, ROESY, and H-detected 1H, 13C and 1H, 31P HMQC experiments, along with chemical methods . The O antigen gene cluster of S . boydii type 13 was located and sequenced . Genes for synthesis of UDP-2-acetamido-2,6-dideoxy-L-glucose and genes that encode putative sugar transferases, O unit flippase, and O antigen polymerase were identified . Seven genes were found to be specific to S . boydii type 13 . The S . boydii type 13 O antigen gene cluster has higher levels of sequence similarity with Vibrio cholerae gene clusters and may be evolutionarily related to these gene clusters.

Int J Food Microbiol, 2004 Jan 15, 90(2), 189 - 95
Survival and growth of acid-adapted and unadapted Shigella flexneri in a traditional fermented Ghanaian weaning food as affected by fortification with cowpea; Tetteh GL et al.; Shigellae are among the major causes of diarrheal diseases in infants and young children in developing countries . We conducted a study to determine the effect of fermentation of corn and corn/cowpea doughs used to prepare a traditional weaning porridge on the survival and growth characteristics of acid-adapted and unadapted Shigella flexneri . Porridges were prepared from doughs fermented for 0, 24, and 48 h at 30 degrees C . Four-strain mixtures of acid-adapted and unadapted S . flexneri cells were separately inoculated (10(4)-10(5) and 10(6)-10(7) cfu/ml) into porridges made from unfermented (pH 5.74-6.05) and fermented (pH 4.07-4.38) doughs . Viability of acid-adapted cells was retained at higher levels in porridge made from fermented dough, compared to unfermented dough, regardless of composition of the porridge or incubation temperature . Cells inoculated into the porridges containing fermented dough were not detected (<1 cfu/ml) within 4 h at 48 degrees C . Results indicate that prior exposure of cells to acid stress renders them more resistant to subsequent acidic conditions . The addition of cowpea flour to corn dough followed by fermentation had little effect on the survival of S . flexneri in porridges made from the dough.

Bioorg Med Chem, 2004 Jan 2, 12(1), 295 - 300
Saturation-transfer difference NMR studies for the epitope mapping of a carbohydrate-mimetic peptide recognized by an anti-carbohydrate antibody; Johnson MA et al.; Saturation-transfer difference NMR spectroscopy (STD-NMR) experiments have been performed to analyze the topography or epitope of the octapeptide MDWNMHAA recognized by the anti-carbohydrate antibody SYA/J6 in solution; the antibody is directed against the Shigella flexneri Y O-antigen polysaccharide . The results permit a valuable comparison of solution versus crystal-structure data, and provide insight for the design of the next-generation binding ligands.

Ned Tijdschr Geneeskd, 2003 Dec 6, 147(49), 2438 - 9
{Shigellosis, a sexually transmitted disease in homosexual men}; Bovee LP et al.; A study of the registered number of Shigella infections in Amsterdam for the period 1997-2001 revealed that in 2001, there was an increase in the number of homosexual patients with oral-anal contacts as source for their infections . All of these infections were caused by subtype Shigella sonnei . The infections were not associated with a specific meeting place for homosexual men . A relationship with an underlying HIV-infection is likely.

Niger Postgrad Med J, 2003 Sep, 10(3), 154 - 6
Multiple drug resistant strains of Shigella isolated in Jos, central Nigeria; Egah DZ et al.; The objective of this study is to highlight the alarming rise in antimicrobial resistance among Shigella species in Jos, Plateau State . Stool samples of eight hundred and ten patients who presented at the Jos University Teaching Hospital with diarrhoea/dysentery were analysed using standard bacteriological techniques . The antimicrobial susceptibility of the isolates were determined . 25 Shigella species were isolated representing 3.1% isolation rate . The male to female ratio is 1.3:1, children aged 0-10 years constituted 16(64%) of the cases . Shigella flexneri (48%) was the most common sero-group . This was followed by S . boydii (24%), then S . sonnei (20%), and S . dysenteriae (8%) . Most strains of shigella species were resistant to Ampicillin (96.0%) . Chloramphenicol (96.0%), Cotrimoxazole (88%), Nalidixic acid (84%) and Tetracycline (75%) . All strains were found to be sensitive to Ciprofloxacin . The drugs of choice in the treatment of Shigella infection in this environment should be Ciprofloxacin and Ofloxacin . Gentamicin was the third drug of choice its use is limited since the infection is not systemic . To avoid continuous abuse of antibiotics in our country there should be an effective legislation by the government to control the indiscriminate purchase of antibiotics.

Comp Immunol Microbiol Infect Dis, 2004 Mar, 27(2), 129 - 39
Serotypes and anti-microbial susceptibility of Plesiomonas shigelloides isolates from humans, animals and aquatic environments in different countries; Gonzalez-Rey C et al.; A total of 73 strains of Plesiomonas shigelloides isolated from humans (24 strains) animals (21 strains) and aquatic environment (28 strains) were determined for their O:H serotype and susceptibility to 18 anti-microbial substances and to the vibriostatic agent O/129 . Of all strains, 86.3% were typeable by the O and 94.5% by the H anti-sera used . The serotype distribution was heterogeneous within a country and between the countries . Of the 57 different serotypes identified, O11:H2 (2 strains), O22:H3 (4 strains), O35:HH11 (2 strains), O52:H3 (2 strains) and O90:H6 (2 strains) were found among isolates from humans and animals (mainly in cats) in Finland and Cuba, and O23:H1a1b (3 strains) among isolates from environmental sources in Slovak Republic and Italy . Most (93-100%) of all strains were susceptible to all anti-microbials tested but resistant (92-96%) to the broad-spectrum penicillins (ampicillin, mezlocillin) . No correlation between anti-microbial resistance patterns and serotypes was found.

Microb Pathog, 2004 Feb, 36(2), 109 - 15
Structure of the Shigella dysenteriae 7 O antigen gene cluster and identification of its antigen specific genes; Feng L et al.; Shigella strains are human pathogens . The O antigen gene cluster of Shigella dysenteriae O7 was sequenced and analyzed . It contains genes for synthesis of nucleotide sugars including UDP-2-acetamido-2-deoxy-D-galacturonamide, UDP-2-acetamido-2-deoxy-D-galacturonic acid and dTDP-4-amino-4,6-dideoxy-D-glucose . Also found in the gene cluster are genes encoding O unit flippase, O antigen polymerase and sugar transferases . The Escherichia coli O121 O antigen, which is present in an important Shiga toxin-producing strain, has the same structure as that of S . dysenteriae O7, and we found that the gene clusters also had the same genes and organization . Four genes specific to S . dysenteriae O7 and E . coli O121 were identified by PCR screening against representatives of 186 E . coli (including Shigella) O serotypes . E . coli O121 and S . dysenteriae O7 isolates can be distinguished by PCR of the H antigen fliC gene.

J Pediatr (Rio J), 1997 Nov-Dec, 73(6), 395 - 400
{Study of in vitro antimicrobial resistance of positive coprocultures to Shigella sp}; dos Santos BA et al.; BACKGROUND: The gastroenteritis caused by Shigella sp (shigellosis) represents an important cause of morbimortality, especially in children under two years old . It is well known that the proper antimicrobial therapy can improve clinical state and diminish the disease dissemination . Increasing levels of resistance to drugs commonly used in the treatment of shigellosis have been described, and its sensibility has not been recently evaluated in our setting . OBJECTIVE: To evaluate the Shigella antimicrobial resistance in our setting . METHODS: A retrospective study was conducted using 106 coprocultures of patients, hospitalized or not, who have been assisted at HCPA and at HPV, during the period of 1994 to 1996, with the sensibility test done through the Kirby-Bauer technique of diffusion discs . RESULTS: Out of the 106 coprocultures studied, 35 (33%) came from HPV and 71 (67%), from HCPA . Considering the species, 58.8% were S . flexneri, 39.6%, S . sonnei, 0.9%, S . disenteriae and 0.9%, S . boydii . The resistance levels to usually indicated antibiotics were: ampicillin -57.5%, trimethropim-sulfamethoxazol -75.5%, and nalidixic acid -7.5% . CONCLUSIONS: These results show significant (p<0.001) in vitro antimicrobial resistance to ampicillin and trimethoprim-sulfamethoxazol and low resistance to nalidixic acid among Shigella sp . Thus, nalidixic acid may be an option to treat infections caused by resistant organisms.

World J Gastroenterol, 2003 Dec, 9(12), 2720 - 5
Identification of alkA gene related to virulence of Shigella flexneri 2a by mutational analysis; Shi ZX et al.; AIM: In vivo induced genes are thought to play an important role during infection of host . AlkA was identified as an in vivo-induced gene by in vivo expression technology (IVET), but its virulence in Shigella flexneri was not reported . The purpose of this study was to identify the role of alkA gene in the pathogenesis of S . flexneri . METHODS: PCR was used to amplify alkA gene of S . flexneri 2a and fragment 028pKm . The fragment was then transformed into 2457T05 strain, a S flexneri 2a strain containing Red recombination system, which was constructed with a recombinant suicide plasmid pXLkd46 . By in vivo homologous recombination, alkA mutants were obtained and verified by PCR and sequencing . Intracellular survival assay and virulence assay were used to test the intracellular survival ability in HeLa cell model and the virulence in mice lung infection model respectively . RESULTS: Deletion mutant of S . flexneri 2a alkA was successfully constructed by gamma Red recombination system . The mutant exhibited significant survival defects and much significant virulence defects in mice infection assay . CONCLUSION: AlkA gene plays an important role in the infection of epithelial cells and is a virulent gene of Shigella spp.

Phytother Res, 2003 Dec, 17(10), 1155 - 7
Cytotoxicity and antimicrobial activity of goniothalamin isolated from Bryonopsis laciniosa; Mosaddik MA et al.; The cytotoxicity and antimicrobial activity of goniothalamin isolated from Bryonopsis laciniosa was investigated . Goniothalamin showed potent cytotoxicity with LC50 values (5.03 microg/mL) comparable with the reference standard agent, gallic acid . It also showed weak antibacterial and significant antifungal activity against a wide range of gram positive and gram negative bacteria and fungi . The antibacterial (minimum inhibitory concentration) effect against Bacillus cereus and Shigella shiga was found to be 64 microg/mL .

Pharmazie, 2003 Nov, 58(11), 824 - 7
The effect of disinfectants on Plesiomonas shigelloides; Hostacka A et al.; The effects of ten commercially available disinfectants on virulence associated properties of Plesiomonas shigelloides were tested . All the disinfectants tested contained quaternary ammonium salts . The majority of the disinfectants when used at subinhibitory concentrations increased surface hydrophobicity as evaluated by bacterial adherence to xylene and decreased bacterial motility in a concentration dependent manner . Disinfectants did not significantly affect lipase activity . However, more than half of the antimicrobials tested increased the resistance of bacteria to hydrogen peroxide . The disinfectants, in a similar manner to antibiotics at concentrations below MIC, interfered with potential virulence factors of Plesiomonas shigelloides.

Appl Environ Microbiol, 2003 Dec, 69(12), 7028 - 31
Shigella dysenteriae type 1-specific bacteriophage from environmental waters in Bangladesh; Faruque SM et al.; Shigella dysenteriae type 1 is the causative agent of the most severe form of bacillary dysentery, which occurs as epidemics in many developing countries . We isolated a bacteriophage from surface water samples from Bangladesh that specifically lyses strains of S . dysenteriae type 1 . This phage, designated SF-9, belongs to the Podoviridae family and has a 41-kb double-stranded DNA genome . Further screening of water samples for the prevalence of the phage revealed 9 of 71 (12.6%) water samples which were positive for the phage . These water samples were also positive in PCR assays for one or more S . dysenteriae type 1-specific genes, including ipaBCD and stx1, and live S . dysenteriae type 1 was isolated from three phage-positive samples . The results of this study suggest that phage SF-9 may have epidemiological applications in tracing the presence of S . dysenteriae type 1 in environmental waters.

Altern Med Rev, 2003 Nov, 8(4), 378 - 94
Bovine colostrums: a review of clinical uses; Kelly GS; Bovine colostrums are the "early" milk produced by cows during the first several days post-parturition . This "early" milk has a nutrient profile and immunological composition that differs substantially from "mature" milk . Included in the nutrient profile are higher amounts of immunoglobulins, growth factors, cytokines, and nucleosides than are found in milk . Bovine colostrums are also rich in oligosaccharides, antimicrobials, and immune-regulating factors . Available evidence suggests a beneficial effect of supplementation of bovine colostrums in improving body composition, aspects of athletic performance, diarrhea in persons with immune-deficiency syndromes, NSAID-induced gastrointestinal disturbances, and aspects of the acute phase response that occurs secondary to surgery . Specific hyperimmune bovine colostrums, produced to have high neutralizing titer activity against Cryptosporidia, H . pylori, measles, rotavirus, and Shigella sp., appear to have clinical utility in conditions associated with these infectious organisms.

Gene, 2003 Dec 11, 322, 85 - 92
Identification and characterization of simple sequence repeats in the genomes of Shigella species; Yang J et al.; A variety of simple sequence repeats (SSRs) have been identified in the genome of Shigella flexneri serotype 2a (strain Sf301), an enteric pathogen that causes bacillary dysentery in man . The distribution of SSRs, with unit length ranging from 1 to 9 nucleotides, was biased in different regions of the genome . The tri-, tetra- and hexanucleotide SSRs prevailed in the coding regions while the mono- and dinucleotide SSRs were more common in the noncoding regions . Many intergenic SSRs are less than 30 bp away from the downstream open reading frames (ORFs), suggesting a potential role in transcriptional regulation . To study polymorphism of SSRs, we compared 17 coding-region SSRs from strain Sf301 with the corresponding sequences from 23 other strains of four Shigella species . Five chromosomal loci were found to be polymorphic, of which those from S . flexneri strains were most variable . Particularly interesting is the C5-1 locus in the coding sequence of the hcaD gene encoding a subunit of ferredoxin reductase . Depending on the insertion of variable numbers of the unit sequence (CGCAG), the Shigella hcaD genes can encode truncated products due to premature stop codons or frame shifts, or products with extended core alpha helices that leads to radical alterations in the predicted tertiary structure . Hence, SSRs may serve as genotyping markers for epidemiological investigations, and may offer insights into evolutionary adaptation of the pathogens.

FEMS Immunol Med Microbiol, 2003 Dec 5, 39(3), 213 - 9
Porin of Shigella dysenteriae enhances mRNA levels for Toll-like receptor 2 and MyD88, up-regulates CD80 of murine macrophage, and induces the release of interleukin-12; Ray A et al.; Sera of patients convalescing from shigellosis reacted strongly and specifically with the 38,000 Da monomer of porin of Shigella dysenteriae type 1 . Since human, the only natural host of S . dysenteriae type 1, recognized the protein through humoral immune response, it is of great significance to study the surface-exposed outer membrane antigen as an adjuvant . Porin treatment of CD11b+ peritoneal cavity (PerC) MPhi of BALB/c mouse was found to up-regulate CD80 on cell surface and had no effect on CD86 expression . The surface expression of CD80 got increased by 1.6-fold in the presence of gamma interferon (IFN-gamma) supporting selective regulation of the B7-1 (CD80) member of the B7 family . MPhi released 7.25 pg of interleukin-12 (IL-12) in the presence of porin . The protein in combination with IFN-gamma augmented profoundly the release of IL-12 by 2.6-fold . Porin-mediated induction of IL-12 release would therefore influence Th1-type response, known to be preferentially triggered due to up-regulation of CD80 expression . Treatment of PerC MPhi by the protein showed an increase of mRNA for both Toll-like receptor (TLR)2 and myeloid differentiation factor 88 (MyD88) by 2- and 2.3-fold respectively, emphasizing that TLR2 is essential for recognition of S . dysenteriae type 1 porin . Understanding the mechanism of adjuvanticity of porin of S . dysenteriae type 1 is a necessary step towards the development of a better adjuvant against shigellosis.

Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 15023 - 8 Epub 2003 Nov 25.
Structural basis of peptide-carbohydrate mimicry in an antibody-combining site; Vyas NK et al.; The structure of a complex between the Fab fragment of the antibody (SYA/J6) specific for the cell surface O-antigen polysaccharide of the pathogen Shigella flexneri Y and an octapeptide (Met-Asp-Trp-Asn-Met-His-Ala-Ala), a functional mimic of the O-antigen, has been determined at 1.8-A resolution . Comparison of the structure with that of the complex with the pentasaccharide antigen {-->2)-alpha-L-Rha-(1-->2)-alpha-L-Rha-(1-->3)-alpha-L-Rha-(1-->3)-beta-D-GlcNAc-(1-->2)-alpha-L-Rha-(1-->} reveals the molecular recognition process by which a peptide mimics a carbohydrate in binding to an antibody . The binding modes of the two ligands differ considerably . Octapeptide binding complements the shape of the combining site groove much better than pentasaccharide binding . Moreover, the peptide makes a much greater number of contacts (126), which are mostly van der Waals interactions, with the Fab than the saccharide (74) . An unusual feature is also the involvement of 12 water molecules in mediating hydrogen bonds between residues within the peptide or of the peptide and Fab . Despite better shape complementarity and greater number of contacts, the octapeptide binds with an affinity (KA = 2.5 x 10(5) M-1, measured by calorimetry) only approximately 2-fold tighter than the pentasaccharide . The structural results are relevant to the design of peptide mimetics with improved affinity for use as vaccines.

Infect Immun, 2003 Dec, 71(12), 7043 - 52
Characterization of an extracellular virulence factor made by group A Streptococcus with homology to the Listeria monocytogenes internalin family of proteins; Reid SD et al.; Leucine-rich repeats (LRR) characterize a diverse array of proteins and function to provide a versatile framework for protein-protein interactions . Importantly, each of the bacterial LRR proteins that have been well described, including those of Listeria monocytogenes, Yersinia pestis, and Shigella flexneri, have been implicated in virulence . Here we describe an 87.4-kDa group A Streptococcus (GAS) protein (designated Slr, for streptococcal leucine-rich) containing 10 1/2 sequential units of a 22-amino-acid C-terminal LRR homologous to the LRR of the L . monocytogenes internalin family of proteins . In addition to the LRR domain, slr encodes a gram-positive signal secretion sequence characteristic of a lipoprotein and a putative N-terminal domain with a repeated histidine triad motif (HxxHxH) . Real-time reverse transcriptase PCR assays indicated that slr is transcribed abundantly in vitro in the exponential phase of growth . Flow cytometry confirmed that Slr was attached to the GAS cell surface . Western immunoblot analysis of sera obtained from 80 patients with invasive infections, noninvasive soft tissue infections, pharyngitis, and rheumatic fever indicated that Slr is produced in vivo . An isogenic mutant strain lacking slr was significantly less virulent in an intraperitoneal mouse model of GAS infection and was significantly more susceptible to phagocytosis by human polymorphonuclear leukocytes . These studies characterize the first GAS LRR protein as an extracellular virulence factor that contributes to pathogenesis and may participate in evasion of the innate host defense.

Infect Immun, 2003 Dec, 71(12), 7002 - 13
Analysis of virulence and inflammatory potential of Shigella flexneri purine biosynthesis mutants; Cersini A et al.; Several Shigella flexneri mutants with defects in aromatic amino acid and/or purine biosynthesis have been evaluated as vaccines in humans or in animal models . To be suitable as a vaccine, a mutant has to show virulence attenuation, minimal reactogenicity, and a good immunogenic potential in animal models . With this aim, we have constructed five S . flexneri 5 (wild-type strain M90T) mutants with inactivation of one or two of the loci purEK, purHD, and guaBA, governing early or late steps of purine biosynthesis . The mutants have been analyzed in vitro in cell cultures and in vivo in the Sereny test and in the murine pulmonary model of shigellosis . M90T guaBA, M90T guaBA purEK, M90T guaBA purHD, and M90T purHD purEK gave a negative result in the Sereny test . In contrast, in the murine pulmonary model all of the strains had the same 50% lethal dose as the wild type, except M90T guaBA purHD, which did not result in death of the animals . Nevertheless, bacterial counts in infected lungs, immunohistochemistry, and reverse transcription-PCR analysis of mRNAs for tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), interleukin-1beta (IL-1beta), IL-6, IL-12, and inducible nitric oxide synthase (iNOS) revealed significant differences among the strains . At 72 h postinfection, M90T guaBA purHD still induced proinflammatory cytokines and factors such as IL-1beta, IL-6, TNF-alpha, and iNOS, along with cytokines such as IL-12 and IFN-gamma . Moreover, in the absence of evident lesions in murine tissues, this mutant highly stimulated major histocompatibility complex class II expression, showing a significant ability to activate the innate immunity of the host.

Antimicrob Agents Chemother, 2003 Dec, 47(12), 3719 - 23
Epidemiology of nalidixic acid resistance and TEM-1- and TEM-52-mediated ampicillin resistance of Shigella sonnei isolates obtained in Korea between 1980 and 2000; Jeong YS et al.; The resistance to ampicillin and nalidixic acid in Shigella sonnei isolates obtained in Korea during the period 1998 to 2000 was characterized . Recently (J . Y . Oh, H . S . Yu, S . K . Kim, S . Y . Seol, D . T . Cho, and J . C . Lee, J . Clin . Microbiol . 41:421-423, 2003) ampicillin and nalidixic acid resistance was found in 49 and 70%, respectively, of the 67 S . sonnei isolates obtained during this period . We analyzed 138 S . sonnei isolates collected during the same period . Ampicillin and nalidixic acid resistance was found in 30 and 86% of the isolates, respectively . The ampicillin resistance was mediated by a TEM-1 beta-lactamase, and TEM-52 extended-spectrum beta-lactamase was identified in one sporadic S . sonnei isolate from 1999 . bla(TEM-1) and bla(TEM-52) were located in conjugative R-plasmids . Tn3 was detected in 41% of the ampicillin-resistant isolates . The R-plasmids from the transconjugants that transferred resistance to ampicillin exhibited different restriction fragment length polymorphism patterns, and a bla(TEM-1) probe was hybridized with the different fragments . The nalidixic acid resistance was exclusively associated with an amino acid substitution, Ser83-->Leu (TCG-->TTG), in gyrA . These findings indicate that the genetically related S . sonnei strains readily acquire resistance to ampicillin, streptomycin, trimethoprim, and sulfamethoxazole but not nalidixic acid through conjugative R-plasmids from difference sources when confronted by antibiotic selective pressures.

FEMS Microbiol Lett, 2003 Nov 21, 228(2), 159 - 66
Shiga toxins and apoptosis; Cherla RP et al.; The enteric pathogens Shigella dysenteriae serotype 1 and Shiga toxin-producing Escherichia coli (STEC) cause bloody diarrheal diseases that may progress to life-threatening extraintestinal complications . Although the S . dysenteriae and STEC differ in the expression of a number of virulence determinants, they share the capacity to produce one or more potent cytotoxins, called Shiga toxins (Stxs) . Following the ingestion of the organisms, the expression of Stxs is critical for the development of vascular lesions in the colon, kidneys and central nervous system . It has been known for some time that following the intracellular routing of Stxs to the endoplasmic reticulum and nuclear membrane, the toxins translocate into the cytoplasm and target ribosomes for damage . However, numerous recent studies have shown that Stxs trigger programmed cell death signaling cascades in intoxicated cells . The mechanisms of apoptosis induction by these toxins are newly emerging, and the data published to date suggest that the toxins may signal apoptosis in different cells types via different mechanisms . Here we review the Stxs and the known mechanistic aspects of Stx-induced apoptosis, and present a model of apoptosis induction.

Microb Drug Resist, 2003, 9 Suppl 1, S17 - 24
Characterization of antimicrobial resistance, plasmids, and gene cassettes in Shigella spp . from patients in vietnam; Iversen J et al.; The objective of the study was to investigate antimicrobial resistance, plasmids and class 1 integrons in 150 Shigella strains isolated from patients with diarrhea in Vietnam . Most isolates were resistant to the majority of antimicrobial agents used for treatment in the isolation areas and 90% were resistant to three or more antibiotics . A total of 20 strains yielded class 1 integrons, which harbored oxa1, dfrA, orfF, and aadA gene cassettes . The most common gene cassette, aadA2, was always located closest to the 3' conserved segment of the integrons and oxa1 and dfrA closest to the 5' end . Plasmid profiles of the 20 class 1 integron-positive strains all contained more than one plasmid, and 14 different profiles were found . No correlation was found between species, antibiograms, plasmid profiles, or presence of class 1 integrons . Conjugation resulted in 25 transconjugants, which all were resistant to four or more antimicrobial agents and all harbored at least one plasmid (>60 kb) . Class 1 integrons were detected in 64% of the transconjugants . Phenotypic resistance pattern and plasmid profiles of the transconjugants seemed independent of the presence of an integron . Class 1 integrons seemed of less importance in phenotypic antibiograms and in transfer of resistance genes than conjugative plasmids.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Nov, 35(11), 1005 - 10
{Co-expression of CFA/I and CS6 of Enterotoxigenic Escherichia coli ( ETEC ) in Shigella flexneri 2a T32 Derivative Strain FWL01}; Zheng JP et al.; Among the known colonization factors of enterotoxigenic Escherichia coli (ETEC), CFA/I and CS6 (the common antigen in the CFA/IV fimbrial antigens ) are two of the most prevalent fimbriae found in clinical isolates but are never expressed by the same wild-type strains . In this study, CFA/I and CS6 of ETEC were co-expressed in Shigella flexneri 2a T32 derivative strain FWL01 by using a host-plasmid lethal balancing system based on asd gene . The results indicate that the recombinant plasmid carrying CFA/I and CS6 could be stably integrated in FWL01 . Expression of the two antigens did not interfere the host growth . The results of immunofluorescence analysis showed that CFA/I and CS6 were localized on the surface of the strain FWL01 . In Balb/c mice orally immunized with the recombinant strain, the immune responses against CFA/I and CS6 were observed . Those observations show the feasibility of a multivalent vaccine expressing different fimbrial antigens in attenuated Shigella flexneri.

Chang Gung Med J, 2003 Aug, 26(8), 611 - 4
Shigella flexneri sepsis in an infant; Yen JB et al.; Shigellosis continues to be an important public health problem in developed countries, since communication in the world village has become more frequent . In addition, this disease is difficult to be prevented because only a small number of bacteria are required to cause infection, and it has exhibited steady trends towards multiple drug resistance . This report describes a 7-month-old female infant with Shigella flexneri sepsis presenting initially with a high fever, watery diarrhea, and dehydration . She was successfully treated with ceftriaxone for 7 days . This case illustrates that Shigella should be included in the differential diagnosis of sepsis associated with diarrhea particularly in young infants traveling to or living in an endemic area . The choice of antimicrobial therapy and the optimal duration for treatment should be carefully evaluated because of the emergence of multidrug-resistant Shigella.

J Clin Microbiol, 2003 Nov, 41(11), 5053 - 8
Temporal shifts in the dominance of serotypes of Shigella dysenteriae from 1999 to 2002 in Dhaka, Bangladesh; Talukder KA et al.; A total of 358 Shigella dysenteriae strains isolated from patients attending the Dhaka treatment center of the International Centre for Diarrheal Disease Research, Bangladesh, between the years 1999 and 2002 were included in this study . S . dysenteriae type 1, the dominant serotype in 1999 (76.4%), declined to 6.5% in 2002 . On the other hand, S . dysenteriae types 2 to 12 were isolated with increasing frequencies of 19, 67, 73.5, and 87% in 1999, 2000, 2001, and 2002, respectively . Of these, types 2 and 4 were the most dominant serotypes, accounting for more than 18.7 and 28.5% of the total isolates, respectively . There was no isolation of serotypes 5, 7, 8, and 13 during this period . Twenty-eight (7.8%) of the isolates were atypical and agglutinated only with the polyvalent antiserum of S . dysenteriae . More than 98% of type 1 strains isolated between 1999 and 2001 were resistant to ampicillin, sulfamethoxazole-trimethoprim, and nalidixic acid . Among other serotypes of S . dysenteriae, Nal(r) type 2 strains were isolated in 2001 and 2002 . Although heterogeneous plasmid profiles were obtained depending on the presence or absence of a single plasmid, core plasmids were defined for particular serotypes . On the other hand, the same plasmid profile was found to be shared by different serotypes . Interestingly, plasmid patterns of types 2 and 4 were almost identical except that a middle-range plasmid of 70 to 60 MDa was present in type 4 in addition to the core plasmids . All the strains harboring the 140-MDa plasmid were positive for the ipaH gene, had Congo red binding abilities, and were positive by the Sereny test, demonstrating their invasive properties.

J Exp Med, 2003 Nov 3, 198(9), 1361 - 8
Mycobacterium marinum escapes from phagosomes and is propelled by actin-based motility; Stamm LM et al.; Mycobacteria are responsible for a number of human and animal diseases and are classical intracellular pathogens, living inside macrophages rather than as free-living organisms during infection . Numerous intracellular pathogens, including Listeria monocytogenes, Shigella flexneri, and Rickettsia rickettsii, exploit the host cytoskeleton by using actin-based motility for cell to cell spread during infection . Here we show that Mycobacterium marinum, a natural pathogen of fish and frogs and an occasional pathogen of humans, is capable of actively inducing actin polymerization within macrophages . M . marinum that polymerized actin were free in the cytoplasm and propelled by actin-based motility into adjacent cells . Immunofluorescence demonstrated the presence of host cytoskeletal proteins, including the Arp2/3 complex and vasodilator-stimulated phosphoprotein, throughout the actin tails . In contrast, Wiskott-Aldrich syndrome protein localized exclusively at the actin-polymerizing pole of M . marinum . These findings show that M . marinum can escape into the cytoplasm of infected macrophages, where it can recruit host cell cytoskeletal factors to induce actin polymerization leading to direct cell to cell spread.

Infect Immun, 2003 Nov, 71(11), 6298 - 306
Comparison of two major forms of the Shigella virulence plasmid pINV: positive selection is a major force driving the divergence; Lan R et al.; All Shigella and enteroinvasive Escherichia coli (EIEC) strains carry a 230-kb virulence plasmid (pINV) which is essential for their invasiveness . There are two sequence forms, pINV A and pINV B, of the plasmid (R . Lan, B . Lumb, D . Ryan, and P . R . Reeves, Infect . Immun . 69:6303-6309, 2001), and the recently sequenced pINV plasmid from Shigella flexneri serotype 5 is a pINV B form . In this study we sequenced the majority of the coding region of the pINV A form from S . flexneri serotype 6 other than insertion sequence or related sequences and compared it with the pINV B form . More than half of the genes sequenced appear to be under positive selection based on their low ratio of synonymous to nonsynonymous substitutions . This high proportion of selected differences indicates that the two pINV forms have functional differences, and comparative studies of pathogenicity in different Shigella-EIEC strains could be informative . There are also genes absent in the S . flexneri serotype 6 plasmid, including the sepA gene encoding serine protease, the major secreted protein of S . flexneri serotype 2a, and the stbAB genes, which encode one of the two partition systems found in S . flexneri serotype 5 . The incompatibility of the two pINV forms appears to be due to either small differences in the mvpAT postsegregational killing system or the presence of an unknown system in pINVA.

Med J Malaysia, 2003 Jun, 58(2), 262 - 7
Species distribution and antibiotic resistance of shigella isolates in an urban community in Malaysia; Lee WS et al.; There is an increasing trend for Shigella isolates worldwide to be resistant to commonly prescribed antibiotics . The species distribution and antibiotic resistance of Shigella species isolated from children in Kuala Lumpur, Malaysia from 1978 to 1997 was reviewed . Three hundred and eighty six isolates were positive for Shigella species, representing 1.4% (95% CI: 1.3%-1.6%) of the 26320 total stool specimens and 13% (95% CI: 11.8%-14.2%) of 2986 isolates positive for bacterial pathogens . Shigella flexneri, constituting 74% of all isolates in the first five years of the study, decreased by 40% during the last five years (95% CI of decrease: 22.1%-57.9%), p-value < 0.0001) to 34% . There was a significant reduction (chi2 for linear trend = 77.6, p-value < 0.001) in the number of Shigella isolates as a percentage of total stool isolates obtained . 58% of the 241 isolates tested for antibiotic sensitivity were resistant to at least one antibiotic, and 42% wEre multi-resistant to three or more antibiotics . Shigella species was not a common pathogen among children admitted with diarrhoea in Kuala Lumpur, and was more likely to be resistant to commonly prescribed antibiotics.

Zh Mikrobiol Epidemiol Immunobiol, 2003 Sep-Oct, (5), 101 - 8
{Specific prophylaxis of shigellosis}; Bondarenko VM; A high level of shigellosis morbidity requires new approaches to the control of bacterial dysentery . According to modern concepts, the outbreaks of Shigella infections are linked both with less intensive epidemic control measures and the objective cyclic character of the epidemic process . In this connection great importance is attached to the development of vaccines for the immunization of high risk groups in territories with unfavorable epidemic conditions and in zones of military conflicts, as well as children of school and pre-school age, who mostly determine seasonal rises of shigellosis morbidity . In this article the data describing new approaches to the creation of new live enteric vaccines on the basis of the knowledge of the genetic control of microbial pathogenicity and the regulation of its expression are presented . Attenuated Shigella mutants, created by different authors and having good prospects to be used for the development of vaccines, are characterized.

J Mol Evol, 2003 Aug, 57(2), 140 - 8
The evolutionary history of Shigella and enteroinvasive Escherichia coli revised; Escobar-Paramo P et al.; In Shigella and enteroinvasive Escherichia coli (EIEC), the etiologic agents of shigellosis in humans, the determinants responsible for entry of bacteria into and dissemination within epithelial cells are encoded by a virulence plasmid . To understand the evolution of the association between the virulence plasmid and the chromosome, we performed a phylogenetic analysis using the sequences of four chromosomal genes (trpA, trpB, pabB, and putP) and three virulence plasmid genes (ipaB, ipaD, and icsA) of a collection of 51 Shigella and EIEC strains . The phylogenetic tree derived from chromosomal genes showed a typical "star" phylogeny, indicating a fast diversification of Shigella and EIEC groups . Phylogenetic groups obtained from the chromosomal and plasmidic genes were similar, suggesting that the virulence plasmid and the chromosome share similar evolutionary histories . The few incongruences between the trees could be attributed to exchanges of fragments of different plasmids and not to the transfer of an entire plasmid . This indicates that the virulence plasmid was not transferred between the different Shigella and EIEC groups . These data support a model of evolution in which the acquisition of the virulence plasmid in an ancestral E . coli strain preceded the diversification by radiation of all Shigella and EIEC groups, which led to highly diversified but highly specialized pathogenic groups.

EMBO J, 2003 Oct 15, 22(20), 5471 - 9
Abl tyrosine kinases are required for infection by Shigella flexneri; Burton EA et al.; Infection by the opportunistic bacterial pathogen Shigella flexneri stimulates tyrosine phosphorylation of host cell proteins, but the kinases involved and their effects on the regulation of cell signaling pathways during bacterial entry remain largely undefined . Here, we demonstrate a requirement for the Abl family of tyrosine kinases during Shigella internalization . Family members Abl and Arg are catalytically activated upon Shigella infection, accumulate at the site of bacterial entry, and are required for efficient bacterial uptake, as internalization is blocked upon targeted deletion of these kinases or treatment with a specific pharmacological inhibitor . We identify the adapter protein Crk as a target for Abl kinases during Shigella uptake, and show that a phosphorylation-deficient Crk mutant significantly inhibits bacterial uptake . Moreover, we define a novel signaling pathway activated during Shigella entry that links Abl kinase phosphorylation of Crk to activation of the Rho family GTPases Rac and Cdc42 . Together, these findings reveal a new role for the Abl kinases, and suggest a novel approach to treatment of Shigella infections through inhibition of host cell signaling pathways.

Cell Microbiol, 2003 Nov, 5(11), 797 - 807
The ShiA protein encoded by the Shigella flexneri SHI-2 pathogenicity island attenuates inflammation; Ingersoll MA et al.; Shigella spp . are the aetiologic agents of dysentery, a severe diarrhoeal syndrome characterized by acute inflammation in the colon . The inflammatory response, which includes recruitment of polymorphonuclear leukocytes (PMN), damages the colonic mucosa and exacerbates the infection . Shigella encodes a pathogenicity island (PAI), SHI-2, which is localized in a region of the chromosome linked to the induction of inflammation . Surprisingly, SHI-2 deletion mutants induce a stronger inflammatory response than wild-type Shigella as measured by increased villus blunting, increased PMN infiltration and induction of apoptosis in a rabbit ileal loop model of shigellosis . Mutational analysis mapped the hyper-inflammatory phenotype to a single gene, shiA . Similar to SHI-2 deletion mutants, infection with a shiA mutant strain induces dramatically elevated levels of inflammation when compared to the wild-type strain . Furthermore, infection with a wild-type strain containing multiple copies of shiA results in fewer infiltrating PMN and apoptotic cells, as well as preservation of a normal villus architecture at the site of infection, thus acting in a dominant fashion over the pro-inflammatory mechanisms of Shigella . The molecular mechanism of action of ShiA is independent of any in vitro phenotype associated with Shigella virulence . Our data suggest that ShiA allows Shigella to attenuate the host inflammatory response in a novel manner.

Enferm Infecc Microbiol Clin, 2003 Oct, 21(8), 401 - 3
{Intercontinental spread of a trimethoprim-resistant strain of Shigella flexneri}; Navia MM et al.; INTRODUCTION: In this study we characterize a trimethoprim-resistant strain of Shigella flexneri recovered from stool samples of an international traveler after a trip to Kenya, and analyze its epidemiological relationship with a set of strains having similar characteristics from Tanzania . METHODS: Clonal relationships were studied by three techniques - plasmid profile, repetitive-element (REP)-PCR and pulse-field gel electrophoresis (PFGE) . The presence of type 1 integrons was studied by PCR and sequencing . The location and transferability of the detected integron was analyzed by conjugation and PCR . RESULTS: The epidemiological studies showed that all six strains studied belonged to the same clone . Furthermore, all of them carried the same gene encoding for trimethoprim resistance (dfrA7), which was located in an integron within a chromosome . CONCLUSION: Continuous epidemiological surveillance is required to control the spread of pathogenic microorganisms and the dissemination of resistance-encoding genes among geographical areas.

Scand J Infect Dis, 2003, 35(8), 503 - 5
CTX-M-3 extended-spectrum beta-lactamase in a Shigella sonnei clinical isolate: first report from Turkey; Acikgoz ZC et al.; A Shigella sonnei strain resistant to cefotaxime and aztreonam (but not ceftazidime) was isolated from the stool sample of a 7-y-old outpatient . Double disk synergy test, isoelectric focusing, polymerase chain reaction and sequence analysis confirmed that the isolate produced CTX-M-3, an extended-spectrum beta-lactamase (ESBL) . This is the first report from Turkey of Shigella spp . producing an ESBL, and of CTX-M type enzyme.

Rev Med Chil, 2003 Jul, 131(7), 727 - 33
{Integrons and antimicrobial resistance gene cassettes in Shigella flexneri strains }; Munoz J et al.; BACKGROUND: The resistance of Shigella flexneri to antimicrobial agents can be associated to the presence of integrons that may contain and express antimicrobial resistance gene cassettes . AIM: To study antimicrobial resistance and the presence of integrons and antimicrobial gene cassettes in Shigella flexneri strains . MATERIAL AND METHODS: In vitro susceptibility to 27 antimicrobials was studied in twenty four Shigella flexneri strains isolated from stools . The presence of integrons class 1, 2 and 3 and antimicrobial resistance gene cassettes was investigated by polymerase chain reaction (PCR) using specific primers for each gene . RESULTS: Most strains were resistant to one of the following antimicrobials: ampicillin, sulphonamide, trimethoprim, tetracycline, streptomycin, sulfamethoxazole-trimethoprim or chloramphenicol . Twenty nine percent were simultaneously resistant to all these antimicrobials . Integrons class 1 and 2 were found in 19 strains (79%) . Class 3 integrons were not found . Gene cassettes dfrA1 and ant(3")I were associated to integrons class 2 in most strains (15/20, 75%) . Genes cat, tetB and blarTEM were detected in 18/24 (75%), 7/24 (29%) and 4/24 (17%) of the strains, respectively and were not associated to any of the studied integrons . Genes that codify enzymes AAC(6')Ib and APH(3')VI were not detected in any strain . CONCLUSIONS: The high frequency of integrons found in the studied strains, could partly explain the increasing antimicrobial resistance of Shigella flexneri strains, isolated in Chile.

Mol Microbiol, 2003 Oct, 50(1), 45 - 60
IcsA, a polarly localized autotransporter with an atypical signal peptide, uses the Sec apparatus for secretion, although the Sec apparatus is circumferentially distributed; Brandon LD et al.; Asymmetric localization of proteins is essential to many biological functions of bacteria . Shigella IcsA, an outer membrane protein, is localized to the old pole of the bacillus, where it mediates assembly of a polarized actin tail during infection of mammalian cells . Actin tail assembly provides the propulsive force for intracellular movement and intercellular dissemination . Localization of IcsA to the pole is independent of the amino-terminal signal peptide (Charles, M., Perez, M., Kobil, J.H., and Goldberg, M.B., 2001, Proc Natl Acad Sci USA 98: 9871-9876) suggesting that IcsA targeting occurs in the bacterial cytoplasm and that its secretion across the cytoplasmic membrane occurs only at the pole . Here, we characterize the mechanism by which IcsA is secreted across the cytoplasmic membrane . We present evidence that IcsA requires the SecA ATPase and the SecYEG membrane channel (translocon) for secretion . Our data suggest that YidC is not required for IcsA secretion . Furthermore, we show that polar localization of IcsA is independent of SecA . Finally, we demonstrate that while IcsA requires the SecYEG translocon for secretion, components of this apparatus are uniformly distributed within the membrane . Based on these data, we propose a model for coordinate polar targeting and secretion of IcsA at the bacterial pole.

Infect Immun, 2003 Oct, 71(10), 5472 - 9
CadC is the preferential target of a convergent evolution driving enteroinvasive Escherichia coli toward a lysine decarboxylase-defective phenotype; Casalino M et al.; Enteroinvasive E . coli (EIEC), like Shigella, is the etiological agent of bacillary dysentery, a particularly severe syndrome in children in developing countries . All EIEC strains share with Shigella the inability to synthesize lysine decarboxylase (the LDC phenotype) . The lack of this function is considered a pathoadaptive mutation whose emergence was necessary to obtain the full expression of invasiveness . Cadaverine, the product of lysine decarboxylation, is a small polyamine which interferes mainly with the inflammatory process induced by dysenteric bacteria . Genes coding for lysine decarboxylase and its transporter constitute a single operon (cadBA) and are expressed at low pH under the positive control of CadC . This regulator is an inner membrane protein that is able to sense pH variation and to respond by transcriptionally activating the cadBA genes . In this study we show that, unlike in Shigella, mutations affecting the cad locus in the EIEC strains we have analyzed are not followed by a novel gene arrangement and that the LCD(-) phenotype is dependent mainly on inactivation of the cadC gene . Introduction of a functional CadC restores cadaverine expression in all EIEC strains harboring either an IS2 element or a defective cadC promoter . Comparative analysis between the cad regions of S . flexneri and EIEC suggests that the LDC(-) phenotype has been attained by different strategies within the E . coli species.

J Health Popul Nutr, 2003 Jun, 21(2), 96 - 102
Prevalence and pattern of antimicrobial resistance of Shigella species among patients with acute diarrhoea in Karaj, Tehran, Iran; MoezArdalan K et al.; The study was carried out to determine the prevalence and pattern of antimicrobial resistance of Shigella species among patients with acute diarrhoea in Karaj, Tehran, Iran . The study included all acute diarrhoea patients who visited the hospitals and treatment centres of Karaj during November 2001-October 2002 . Of 734 stool samples collected from patients with acute diarrhoea and analyzed for Shigella spp., 123 (16.8%) yielded Shigella spp . (7.5% Shigella flexneri, 5.2% S . sonnei, 2.6% S . dysenteriae, and 1.5% S . boydii) . Of the Shigella isolates, 90.8% were resistant to one or more antimicrobial agent(s), and 87.8% were multidrug resistant . The most common resistance was to tetracycline (73.5%), trimethoprim-sulphamethoxazole (70.4%), and amoxicillin-clavulanic acid (50.0%) . Resistance to cefixime, ciprofloxacin, ceftriaxone, and nalidixic acid was observed in 6.1%, 3.1%, 2.0%, and 1.0% of the isolates respectively . These findings suggest that Shigella spp . may be an important aetiological agent of diarrhoea with a high rate of drug resistance in this region, which requires further study.

J Health Popul Nutr, 2003 Jun, 21(2), 90 - 5
Patterns and properties of haemagglutinins expressed by Shigella serogroups in Lagos, Nigeria; Iwalokun BA et al.; Forty-five strains of Shigella were screened for haemagglutinin production and broad-spectrum haemagglutination reaction . Mannose-sensitive haemagglutinin (MSHA) was found in 22 strains {Shigella flexneri (7), S . dysenteriae (7), S . sonnei (3), and S . boydii (5)} . Eighteen strains harboured mannose-resistant haemagglutinin (MRHA), and 8 strains were observed to be non-haemagglutinating to guinea pig erythrocyte . With the exception of human erythrocytes (O, A, B, and AB), the observed MSHA and MRHA also agglutinated the erythrocytes of rabbit, sheep, rat, chicken, and horse, suggesting a broad-spectrum haemagglutinating property . Haemagglutinins of S . flexneri and S . dysenteriae elicited a relatively stronger haemagglutinating activity with agglutinability to chicken and rabbit erythrocytes enhanced by trypsinization . Haemagglutination reaction with guinea pig erythrocyte was generally inhibited by sialic acid, while simple sugars, such as D-glucose, D-galactose, N-acetylgalactosamine, N-acetylglucosamine, and D-rhamnose, elicited no inhibitory effect . The results of the study revealed broad-spectrum haemagglutinin expression by circulating Shigella strains in Nigeria.

J Clin Microbiol, 2003 Sep, 41(9), 4238 - 45
Cohort study of Guinean children: incidence, pathogenicity, conferred protection, and attributable risk for enteropathogens during the first 2 years of life; Valentiner-Branth P et al.; We recruited 200 children shortly after birth and collected stool specimens weekly, irrespective of whether the children had diarrhea, until up to 2 years of age . All children were recruited during the first year of the study and were monitored for a median of 18.4 months . To measure pathogenicity, the odds ratio for diarrhea, adjusted for age, sex, and coinfections with other enteropathogens, was determined by logistic regression . Standard estimation of the population attributable risk indicated that rotavirus, enterotoxigenic Escherichia coli that produced only the heat-stable toxin ST, Isospora spp., Cryptosporidium parvum, Shiga toxin (Stx)-producing E . coli (STEC), and Shigella spp . or enteroinvasive E . coli were the most important contributors to diarrhea in this population . Stx2- but not Stx1-producing STEC strains were pathogenic . Enteroaggregative E . coli, diffusely adherent E . coli, and attaching-and-effacing E . coli strains, which were the most commonly isolated microorganisms, were not associated with diarrhea . For most of the microorganisms, primary infections did not confer protection against reinfection with the same organism, but some conferred protection against diarrhea from reinfection.

Lancet, 2003 Aug 30, 362(9385), 705 - 6
Shigella dysenteriae serotype 1 in west Africa: intervention strategy for an outbreak in Sierra Leone; Guerin PJ et al.; In November 1999, a Medecins Sans Frontieres team based in the southeastern part of Sierra Leone reported an increased number of cases of bloody diarrhoea . Shigella dysenteriae serotype 1 (Sd1) was isolated in the early cases . A total of 4218 cases of dysentery were reported in Kenema district from December, 1999, to March, 2000 . The overall attack rate was 7.5% . The attack rate was higher among children younger than 5 years than in the rest of the population (11.2% vs 6.8%; relative risk=1.6; 95% CI 1.5-1.8) . The case fatality was 3.1%, also higher for children younger than 5 years (6.1% vs 2.1%; relative risk=2.9; 95% CI 2.1-4.1}) . Among 583 patients regarded at increased risk of death who were treated with ciprofloxacin in isolation centres, case fatality was 0.9% . A 5-day ciprofloxacin regimen, targeted to the most severe cases of bloody diarrhoea, was highly effective . This is the first time a large outbreak caused by Sd1 has been reported in west Africa.

Microbiology, 2003 Sep, 149(Pt 9), 2609 - 14
Human microvascular endothelial cells resist Shiga toxins by IFN-gamma treatment in vitro; Yoshida T et al.; Shiga toxins (Stxs) produced by enterohaemorrhagic Escherichia coli or Shigella dysenteriae damage human endothelial cells predominantly in cooperation with pro-inflammatory cytokines, such as TNF-alpha . However, in this study, in vitro IFN-gamma pre-treatment resulted in human lung microvascular endothelial cells becoming over 10,000-fold less sensitive to Stxs . In contrast, in their basal condition, they were extremely sensitive to Stxs . Interestingly, TNF-alpha addition to IFN-gamma reverted the Stx-resistant phenotype, which corresponded with its well-established enhancing effect on Stx toxicity . Toxin binding to the cell was barely affected by IFN-gamma . Also, the toxin uptake in the Stx-resistant phenotype was more than 100-fold greater than that of normal cells, when compared at Stx concentrations resulting in equivalent degrees of cell damage . Protein synthesis was inhibited by nearly 90% in the Stx-resistant phenotype after 24 h toxin exposure . This indicated that the intracellular toxin was active as an N-glycosidase, while cells were still over 60% viable, suggesting a possible unknown cytotoxic function of Stx . In conclusion, this study shows a unique effect of IFN-gamma in the suppression of the toxicity of Stxs in a human microvascular endothelial cell model and the involvement of a novel mechanism in this suppression.

Microbiology, 2003 Sep, 149(Pt 9), 2513 - 27
Shigella-induced necrosis and apoptosis of U937 cells and J774 macrophages; Nonaka T et al.; It is currently unclear whether Shigella kills its phagocytic host cells by apoptosis or necrosis . This study shows that rapid necrosis ensues in macrophage-like cell lines (U937 cells differentiated by all-trans-retinoic acid and J774 cells) infected with the Shigella flexneri strain YSH6000 . The infected cells rapidly lose membrane integrity, a typical feature of necrosis, as indicated by the release of the cytoplasmic lactate dehydrogenase and the exposure of phosphatidylserine (PS) associated with the rapid uptake of propidium iodide (PI) . The infected cells exhibit DNA fragmentation without nuclear condensation, and substantial involvement of either caspase-3/-7 or caspase-1 was not detected, which is also contrary to what is normally observed in apoptosis . Cytochalasin D potently inhibited Shigella-induced cell death, indicating that only internalized Shigella can cause necrosis . Osmoprotectants such as polyethylene glycols could suppress cell death, suggesting that insertion of a pore by Shigella into the host cell membrane induces the necrosis . The pore was estimated to be 2.87+/-0.4 nm in diameter . Shigella was also found to be able to induce apoptosis but only in one of the lines tested and under specific conditions, namely U937 cells differentiated with interferon-gamma (U937IFN) . Caspase-3/-7 but not caspase-1 activation was observed in these infected cells and the exposure of PS occurred without the uptake of PI . An avirulent Shigella strain, wild-type Shigella killed with gentamicin, and even Escherichia coli strain JM109, could also induce apoptosis in U937IFN cells, and cytochalasin D could not prevent apoptosis . It appears therefore that Shigella-induced apoptosis of U937IFN cells is unrelated to Shigella pathogenicity and does not require bacterial internalization . Thus, Shigella can induce rapid necrosis of macrophage-like cells in a virulence-related manner by forming pores in the host cell membrane while some cells can be killed through apoptosis in a virulence-independent fashion.

Gig Sanit, 2003 Jul-Aug, (4), 6 - 8
{Acute intestinal infections in children in areas with industrial environmental air pollution}; Savilov ED et al.; Acute intestinal infections were clinically and epidemiologically studied in children residing in the towns with different quantitative and qualitative composition of ambient air pollutants and in the districts of a town, which differ in the level of technogenic ambient air pollution . Six hundred and eighty patients with different types of shigellosis and 421 patients with salmonellosis admitted to the infection hospitals of the towns of Angarsk (an intensively polluted locality) and Irkutsk (a better ecological area) were examined in 1995 to 2000 . The technogenic ambient air pollution was found to exert a noticeable impact on the incidence with S . sonnei dysentery . In poor environmental areas, all the infections under study are characterized by a great burden, duration, more severe clinical symptoms, and poor laboratory changes in the presence of a decreased responsiveness.

Plasmid, 2003 Sep, 50(2), 161 - 7
Use of endogenous host plasmids for generation of Escherichia coli O157:H7 and Shigella sonnei strains that stably express the green fluorescent protein; Monday SR et al.; The gfp gene was manipulated from a commercially available, high copy vector into endogenous plasmids of Escherichia coli O157:H7 and Shigella sonnei to yield stable GFP strains that required neither high copy number for visualization nor antibiotics for stable maintenance of the phenotype . The GFP phenotype of these strains remained stable after repeated passages in media and conditions that enhance plasmid instability and loss from bacterial cells . These results demonstrate the utility of the endogenous plasmids in selectively marking bacteria without altering host cellular function or biochemical properties.

Electrophoresis, 2003 Aug, 24(16), 2864 - 82
A two-dimensional proteome map of Shigella flexneri; Liao X et al.; Shigella flexneri is a Gram-negative facultatively intracellular pathogen responsible for bacillary dysentery in humans . In this study, extracellular proteins from the culture medium and whole cell proteins in cellular extracts of S . flexneri 2a strain 2457T were examined by two-dimensional (2-D) gel electrophoresis using immobilized pH gradient (IPG) technology . Proteins were identified by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with Mascot search program . In total, among the 488 proteins spots processed, 388 proteins were identified . The identified proteins represented 169 genes . By comparing results of Mascot search against databases of Escherichia coli and genomes of S . flexneri 2a, one S . flexneri-specific protein was identified and one possible gap was found in 2457T genome sequences . Although this proteome map is still incomplete, it is already a useful reference for future studies involving pathogenicity, vaccine development, design of novel antibacterial drugs, etc . Proteome maps and a table of all identified proteins are available on the internet at www.proteomics.com.cn.

Int J Food Microbiol, 2003 Oct 15, 87(1-2), 131 - 8
Survival, growth, and inactivation of acid-stressed Shigella flexneri as affected by pH and temperature; Tetteh GL et al.; A study was done to determine the survival, growth, and inactivation characteristics of unadapted, acid-adapted, and acid-shocked Shigella flexneri 2a cells as affected by pH and temperature . The pathogen was grown at 37 degrees C for 18 h in tryptic soy broth containing no glucose (TSBNG) (unadapted cells) and TSBNG supplemented with 1% glucose (TSBG) (acid-adapted cells) . Cells grown in TSBNG were acid-shocked by adjusting 18-h cultures to pH 4.5+/-0.05 with lactic acid . All three cell types were separately inoculated into tryptic soy broth (6.6-7.0 log(10) cfu/ml) containing 0.25% glucose (TSB) acidified to pH 3.5-5.5 with lactic acid and incubated at 4, 12, 21, 30, and 48 degrees C for up to 144 h . Overall, inactivation of S . flexneri cells at low pH was enhanced with an increase in incubation temperature . All three types of cells survived for 144 h at 4 degrees C in TSB acidified to pH 3.5, compared to < 24 h at 30 degrees C and 2 h at 48 degrees C . The population of all three cell types increased significantly (alpha = 0.05) within 24 h when cells were incubated at 12, 21, or 30 degrees C in TSB at pH 5.0, 5.5, or 7.3 . Prior exposure of the S . flexneri to an acidic environment (acid-adapted or acid-shocked cells) resulted in increased resistance to extreme acid and temperature conditions . Acid-adapted cells decreased by approximately 2.5 log(10) cfu/ml when incubated at 4 degrees C for 144 h, compared to a 6-log(10) reduction in control (unadapted) cells . When cells were exposed to low pH (3.5-4.5) and high temperature (48 degrees C), significantly higher (alpha = 0.05) populations were recovered on tryptic soy agar (TSA) than on TSA supplemented with 4% NaCl (TSAS), indicating that a portion of S . flexneri cells were injured . Results show that the ability of S . flexneri to survive and grow at a given pH is influenced by previous exposure to acidic environments and by incubation temperature.

Microb Pathog, 2003 Sep, 35(3), 107 - 17
IpaC of Shigella binds to the C-terminal domain of beta-catenin; Shaikh N et al.; IpaC of Shigella is essential for initial bacterial entry into epithelial cells . We report here that IpaC interacts with beta-catenin and destabilizes the cadherin-mediated cell adhesion complex . Using a yeast two-hybrid system, we identified beta-catenin as a binding partner of IpaC within the host cell after cell entry, but not in the initial entry . Co-immunoprecipitation, confocal microscopy, and GST pull-down experiments confirmed the intracellular and cell-free interactions between these two proteins . The interaction sites were mapped to the ninth armadillo repeat of beta-catenin and to the C-terminus of IpaC . IpaC-associated beta-catenin was phosphorylated at tyrosine residues . This phosphorylation led to the destabilization of the functional cadherin-catenin complex, which could be a mechanism whereby the epithelial cell-cell tight adhesion is disrupted . These events may facilitate the further basolateral invasion of bacteria through the disrupted space and/or modulate the cell-to-cell spread of Shigella.

Przegl Epidemiol, 2003, 57(1), 77 - 84
{Dysentery in Poland in 2001}; Stypulkowska-Misiurewicz H et al.; The notified number of dysentery cases is still very low, similar to the number reported in 2000 . Only 128 cases were notified in the year 2001 (incidence 0.33/100,000 population) while 121 cases were notified in 2000 (incidence 0.31/100,000) . No death due to dysentery was notified . The last three death cases were notified in 1999 . Only one outbreak of foodborne infection due to S . sonnei involving 28 patients was registered, less than in 2000 when 2 outbreaks, one due to S . flexneri and one due to S . sonnei, and less than in 1999, when four outbreaks involving 146 patients were registered . S . sonnei remains the dominant etiological agent . It was isolated in 91% of notified dysentery cases and S . flexneri was isolated only in 10% of cases . S . dysenteriae and S . boydii were not found among persons examined bacteriologically by laboratory service of Epidemiological and Sanitary Stations . The external control of selective media used in 37 laboratories for routine bacteriological diagnosis of humans intestinal infections in the Sanitary--Epidemiological Stations (SSE) in Poland was performed . It was shown that the quality of the Mac Conkey medium was adequate for all 4 bacterial strains checked: S . sonnei, S . flexneri, S . Typhi and S . Enteritidis . All specimens of the SS medium were acceptable only for diagnosis of S . Enteritidis infections . The quality of media used in nearly 1/3 of SSE bacteriological laboratories was inadequate for diagnosis of S . flexneri infections and in some laboratories even not sufficient for diagnosis of infections due to S . Typhi and S . sonnei . In the period of low frequency of Shigella infections the external control of the quality of bacteriological media and laboratory procedures is needed and should be done regularly.

J Biol Chem, 2003 Nov 28, 278(48), 47928 - 36 Epub 2003 Aug 18.
Conformational studies of the O-specific polysaccharide of Shigella flexneri 5a and of four related synthetic pentasaccharide fragments using NMR and molecular modeling; Clement MJ et al.; As part of a program for the development of synthetic vaccines against the pathogen Shigella flexneri, we used a combination of NMR and molecular modeling methods to study the conformations of the O-specific polysaccharide (O-SP) of S . flexneri 5a and of four related synthetic pentasaccharide fragments . The NMR study, based on the analysis of 1H and 13C chemical shifts, the evaluation of inter-residue distances, and the measurement of one- and three-bond heteronuclear coupling constants, showed that the conformation of one of the four pentasaccharides is similar to that of the native O-SP in solution . Interestingly, inhibition enzyme-linked immunosorbent assay demonstrated that a protective monoclonal antibody specific for S . flexneri 5a has a greater affinity for this pentasaccharide than for the others . We carried out a complete conformational search on the pentasaccharides using the CICADA algorithm interfaced with MM3 force field . We calculated Boltzmann-averaged inter-residue distances and 3JC,H coupling constants for the different conformational families and compared the results with NMR data for all pentasaccharides . Our experimental data are consistent with only one conformational family . We also used molecular modeling data to build models of the O-SP with the molecular builder program POLYS . The models that are in agreement with NMR data adopt right-handed 3-fold helical structures in which the branched glucosyl residue points outwards.

J Bacteriol, 2003 Sep, 185(17), 5158 - 65
Determination of the InvE binding site required for expression of IpaB of the Shigella sonnei virulence plasmid: involvement of a ParB boxA-like sequence; Taniya T et al.; The InvE protein positively regulates the expression of virulence genes ipaBCD in Shigella sonnei . The InvE has significant homology with ParB of plasmid P1, which is known as a plasmid partitioning factor with DNA binding ability . Although the DNA binding activity of InvE has been predicted, it is not known whether the DNA binding activity is necessary for type III secretion system-associated gene expression . In this study, we determined the transcription start site of the icsB-ipaBCD operon (ipa operon) and constructed a series of deletions of the icsB promoter region in the Escherichia coli K-12 background . The deletion study revealed that an 86-bp region upstream of the icsB transcription start site was essential for expression of the ipa operon, where the ParB binding motif (ParB BoxA-like sequence) was observed . Purified glutathione S-transferase-InvE fusion protein bound directly to the -93 to -54 region (designating the icsB transcription start site as nucleotide +1) containing the ParB BoxA-like sequence . These results indicated that InvE bound directly to the promoter region.

Acta Microbiol Pol, 2003, 52(1), 45 - 52
The factor C3 conversion in human complement by smooth Shigella flexneri lipopolysaccharides; Fudala R et al.; 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.

Pediatr Infect Dis J, 2003 Aug, 22(8), 701 - 6
Safety and immunogenicity of Shigella sonnei-CRM9 and Shigella flexneri type 2a-rEPAsucc conjugate vaccines in one- to four-year-old children; Passwell JH et al.; BACKGROUND AND OBJECTIVE: Shigella conjugate vaccines have been shown to be safe, immunogenic and efficacious in adult volunteers . We have now investigated the safety and immunogenicity of investigational Shigella sonnei and Shigella flexneri 2a conjugate vaccines in 1- to 4-year-old children, the age group at greatest risk for shigellosis . METHODS: The O-specific polysaccharides of S . sonnei and S . flexneri 2a, the two most common shigellae from patients in Israel, were bound to medically useful carrier proteins to form conjugates . Eighty healthy 1- to 4-year-olds were randomized to receive two 0.5-ml im injections 6 weeks apart of either S . sonnei-CRM(9) or S . flexneri 2a-rEPA(succ) . Blood was taken before, 6 weeks after the first injection, 4 weeks after the second injections and 2 years after immunization for assay of IgG anti-lipopolysaccharide, diphtheria toxin and Pseudomonas aeruginosa exotoxin A antibodies by enzyme-linked immunosorbent assay . RESULTS: During an 8-day surveillance period after each immunization, low fever (37.8-39.0 degrees C) lasting only 24 to 48 h occurred in 2 of 40 recipients after the first injection and 4 of 40 recipients after the second injection of S . flexneri 2a-rEPA(succ) and in 2 of 38 of S . sonnei-CRM(9) after the second injection; no fever was detected after the first injection . Liver function tests were normal in all vaccinees . S . sonnei-CRM(9) elicited a >4-fold rise in IgG anti-LPS in 92.1% and S . flexneri 2a-rEPA(succ) in 85% (P < 0.0001) after the second injection; both conjugates elicited type-specific booster responses . At 2 years the geometric mean concentrations of both IgG anti-lipopolysaccharides were significantly higher than preimmunization levels . A >4-fold rise of IgG anti-diphtheria (65.8%) and IgG anti-ETA (77.5%) was observed . CONCLUSION: These experimental Shigella conjugate vaccines were safe and immunogenic in 1- to 4-year-old children.

FEMS Microbiol Lett, 2003 Aug 8, 225(1), 155 - 60
Identification of the promoter regions and sigma(s)-dependent regulation of the gadA and gadBC genes associated with glutamate-dependent acid resistance in Shigella flexneri; Waterman SR et al.; Resistance to killing by low pH is a common feature of both Escherichia coli and Shigella flexneri . The most effective E . coli acid resistance system utilizes two isoforms of glutamate decarboxylase encoded by gadA and gadB, and a putative glutamate/gamma-amino butyric acid antiporter encoded by gadC . Expression of the gad system is dependent upon the alternate sigma factor, sigma(s) . We confirm that gadA, gadB, and gadC are also all dependent upon sigma(s) for their expression in S . flexneri . -10 sequences similar to the sigma(s)-10 consensus sequence were identified by primer extension in the upstream promoters of all three genes and the transcriptional start points were identical in both E . coli and S . flexneri.

Biochemistry, 2003 Aug 12, 42(31), 9498 - 506
Thermodynamic analysis of the structural stability of the shiga toxin B-subunit; Pina DG et al.; The conformational stability of Shiga toxin B-subunit (STxB), a pentameric protein from Shigella dysenteriae has been characterized by high sensitivity differential scanning calorimetry and circular dichroism spectroscopy under different solvent conditions . It is shown that the thermal folding/unfolding of STxB is a reversible process involving a highly cooperative transition between folded pentamer and unfolded monomers . The conformational stability of STxB is pH dependent and because of its pentameric nature is also concentration dependent . STxB is maximally stable in the pH range from 5 to 9 (Delta G upon unfolding is close to 13 kcal per mol of monomer at 25 degrees C), and its stability decreases both at lower and at higher pH values . The pH dependence of the Gibbs energy of stabilization between pH 2.5 and 5 is consistent with the change in the ionizable state of an average of four groups per monomer upon unfolding . Structural thermodynamic calculations show that the stabilization of the STxB pentamer is primarily due to the interactions established between monomers rather than intramonomer interactions . The folding of an isolated monomer into the conformation existing in the pentamer is unfavorable and expected to be characterized by a free-energy change upon folding in the order of 2.5 kcal mol(-1) at 25 degrees C . On the average, intersubunit interaction induced upon oligomerization of folded monomers should contribute close to -13.4 kcal per mol of monomer to bring the overall Gibbs energy to the experimentally determined value at this temperature.

Mol Genet Genomics, 2003 Oct, 270(1), 66 - 77 Epub 2003 Aug 01.
Shigella flexneri 2a strain 2457T expresses three members of the H-NS-like protein family: characterization of the Sfh protein; Beloin C et al.; Shigella flexneri 2a is known to express the H-NS nucleoid-structuring protein and the paralogous protein StpA . Using bioinformatic analysis we have now discovered a third member of the H-NS protein family, Sfh (Shigella flexneri H-NS-like protein), in strain 2457T . This protein is encoded by the sfh gene, which is located on a high-molecular-mass plasmid that is closely related to the self-transmissible plasmid R27 . When expressed in Escherichia coli, the Sfh protein can complement an hns null mutation, restoring wild-type Bgl, porin protein, and mucoidy phenotypes, and wild-type expression of the fliC and proU genes . While a knockout mutation in the sfh gene alone had no effect on the expression of virulence genes in S . flexneri, an additive effect on virulence gene derepression was seen when the sfh lesion was combined with a mutation in hns . Over-expression of the sfh gene repressed expression of the VirB virulence regulatory protein and transcription of a VirB-dependent structural gene promoter . The purified Sfh protein bound specifically to DNA sequences containing the promoters of the virF and virB virulence regulatory genes . These findings show that Sfh has the ability to influence genetic events beyond the genetic element that encodes it, including the expression of the S . flexneri virulence genes . They raise the possibility of a triangular relationship among three closely related proteins with broad consequences for genetic events in the bacterium that harbours them.

FEMS Microbiol Lett, 2003 Jul 29, 224(2), 277 - 83
Conversion of Shigella flexneri serotype 2a to serotype Y in a shigellosis patient due to a single amino acid substitution in the protein product of the bacterial glucosyltransferase gtrII gene; Chen JH et al.; Conversion of serotype from 2a to Y was demonstrated with five Shigella flexneri isolates recovered from an infected patient . When introduced into the serotype Y isolate, the glucosyltransferase (gtr) II gene of the serotype 2a isolate is capable of inducing the conversion from serotype Y to 2a . In contrast, the gtrII of the serotype Y isolate lacks the capacity to change serotype, resulting from a Cys-->Tyr substitution in its predicted protein sequence . The protein product of the gtrII gene was detected . This is the first report of serotype conversion of S . flexneri in humans, and successful detection of the protein product from a gtr gene.

Zh Mikrobiol Epidemiol Immunobiol, 2003 May-Jun, (3), 50 - 4
{Comparative information value of humoral immunity characteristics in some bacterial and viral infections}; Saidova BM et al.; Serological examination of 144 patients with different bacterial and viral infections was carried out . Antibodies to Brucella were detected in blood serum in 42 patients (85.7%) with the average titer of 1:996 and in saliva in 41 patients (83.7%) with the average titer of 1:567 by passive hemagglutination test with brucella erythrocyte diagnosticum . Out of 26 dysentery patients, antibodies in blood serum were detected in the diagnostic titer in 17 patients (65.4%) with the average titer of 1:282 and in saliva in 21 patients (80.8%) in the titer of 1:100 and higher . Anti-HAV and anti-HBc IgM were detected in specimens of saliva from patients with serologically confirmed viral hepatitis A and B in 100% of cases . The presence of HBsAg in saliva from hepatitis B patients was established in 95.4% of cases . In blood serum and in specimens of saliva anti-HCV IgM were detected in 100% and 85.7% of cases respectively . Out of 25 women with aggravated obstetric history, IgG antibodies to CMV were detected in blood serum in 23 women (88.5%) and in saliva in 22 women (84.6%) . The results of these investigations revealed that the detection rate of antibodies in blood serum and saliva in cases of infections, both bacterial (brucellosis, shigellosis) and viral (hepatitis A, B, C and CMV infection), was not essentially different . The simplicity of obtaining material for analysis make it possible to recommend the use of saliva for diagnosing bacterial and viral infections, especially in mass epidemiological surveys.

Bioorg Chem, 2003 Aug, 31(4), 331 - 44
tRNA-guanine transglycosylase from E . coli: a ping-pong kinetic mechanism is consistent with nucleophilic catalysis; Goodenough-Lashua DM et al.; tRNA-guanine transglycosylase (TGT) is a key enzyme in the post-transcriptional modification of certain tRNAs with the pyrrolopyrimidine base queuine . TGT is required for pathogenicity in Shigella flexneri, a human pathogen, and therefore is potentially a novel antibacterial target . Previous work has indicated that the TGT reaction proceeds through a covalent enzyme-tRNA complex {Biochemistry 40 (2001) 14123} . To further substantiate this mechanism, the determination of the kinetic mechanism for the TGT reaction was undertaken . Computational and graphical analyses of initial velocity data are most consistent with a ping-pong kinetic mechanism . The modes of inhibition of 7-methylguanine with respect to both guanine (competitive) and tRNA (uncompetitive) indicate that tRNA binds first to the enzyme . This kinetic mechanism is consistent with the covalent intermediate chemical mechanism and with our earlier study of a mechanism-based inhibitor {7-fluoromethyl-7-deazaguanine, Biochemistry 34 (1995) 15539} in which TGT inactivation was dependent upon the presence of tRNA.

Expert Opin Pharmacother, 2003 Aug, 4(8), 1315 - 20
An evaluation of current shigellosis treatment; Bhattacharya SK et al.; Shigellosis or bacillary dysentery, characterised by the passage of frequent loose stools mixed with blood and mucous, is caused by Shigella spp . which can be subdivided into four serotypes, namely, S . sonnei, S . boydii, S . flexneri and S . dysenteriae . S . dysenteriae type 1 produces severe dysentery and may be associated with many complications like leukaemoid reaction and haemolytic-ureamic syndrome . It is known to produce protracted epidemics and pandemics and is usually multi-drug resistant . Antibiotics are the mainstay of therapy of all cases of shigellosis . Antibiotics such as tetracycline, ampicillin and co-trimoxazole, were previously highly effective . Newer fluoroquinolones such as norfloxacin, ciprofloxacin, ofloxacin, azithromycin and ceftriaxone are effective . Although single dose of norfloxacin 800 mg and ciprofloxacin 1 g have been shown to be effective, they are currently less effective against S . dysenteriae type 1 infection . Oral rehydration salt should be given concurrently to prevent or correct dehydration . Antimotility agents are contraindicated . Feeding during and after shigellosis is emphasised . Hand-washing practices with plenty of water and soap help to prevent the transmission of infection from person to person . A search is on for an effective vaccine against shigella.

Mol Microbiol, 2003 Aug, 49(3), 755 - 67
MxiK and MxiN interact with the Spa47 ATPase and are required for transit of the needle components MxiH and MxiI, but not of Ipa proteins, through the type III secretion apparatus of Shigella flexneri; Jouihri N et al.; The type III secretion (TTS) pathway is used by numerous Gram-negative pathogens to inject virulence factors into eukaryotic cells . The Shigella flexneri TTS apparatus (TTSA) spans the bacterial envelope and its assembly requires the products of approximately 20 mxi and spa genes . We present a functional analysis of the mxiK, mxiN and mxiL genes . Inactivation of mxiK and mxiN, but not mxiL, resulted in the assembly of a non-functional TTSA that lacked the outer needle . The amounts of needle components MxiH and MxiI were drastically reduced in mxiK and mxiN mutants and in the secretion defective spa47 mutant, indicating that MxiH and MxiI are degraded if they do not transit through the TTSA . Remarkably, expression of MxiH-His in the mxiN mutant and MxiI-His in the mxiK mutant restored assembly of a functional TTSA, as shown by the ability of these strains to enter into epithelial cells and to secrete Ipa proteins in response to activation by Congo red . Using a two-hybrid screen in yeast and immunoprecipitation assays from S . flexneri extracts, we identified interactions between MxiK and Spa33 and Spa47 and between MxiN and Spa33 and Spa47 . These results suggest that transit of the needle components MxiH and MxiI through the TTSA involves the concerted action of the cytoplasmic proteins Spa47, Spa33, MxiK and MxiN . They also show that neither MxiK nor MxiN are absolutely required for secretion of Ipa proteins, provided that the TTSA is correctly assembled.

Clin Pediatr (Phila), 2003 Jun, 42(5), 411 - 5
Shigella bacteremia: a retrospective study; Greenberg D et al.; The aims of this study were to determine the risk factors in, and the clinical and laboratory characterizations of, Shigella bacteremia, as well as the subspecies of Shigella, and the antibiotic susceptibility . A retrospective study of all patients younger than 18 years of age with documented Shigella bacteremia from January 1989 through December 2001 was conducted . Fifteen children with Shigella bacteremia were treated at our center . The mean age (+/- SD) was 20.5 months (+/- 34.2), median 7 months . Thirteen (87%) patients failed to gain weight . The mean duration of diarrhea was 14.7 days . Patients were hospitalized for a mean (+/- SD) of 13.5 days (+/- 9.2) . There were no fatalities in our study sample . The vast majority (86.7%) of the Shigella isolates were flexneri . Most isolates were susceptible to ceftriaxone, ciprofloxacine, and gentamicin but resistant to ampicillin and trimethoprim/sulfamethoxazole.

Ann N Y Acad Sci, 2003 Jun, 990, 535 - 47
Rickettsial actin-based motility: behavior and involvement of cytoskeletal regulators; Heinzen RA; Actin-based motility (ABM) is employed by spotted fever group (SFG) rickettsiae, such as Rickettsia rickettsii, to promote cell-to-cell spread . Time-lapse video microscopy revealed that ABM is not strictly confined to SFG rickettsiae as typhus group R . typhi moved at approximately the same rate as R . rickettsii (approximately 4 micro m/min), but in a highly erratic fashion . A number of common behaviors were observed between ABM of R . typhi and R . rickettsii, such as entrance into plasma membrane protrusions, formation of new actin tails only on the old surface of newly formed daughter cells, and quick (within 15 sec) reassembly of the actin tail to the opposite pole upon contact with cellular structures that impede forward movement . This last behavior suggests that the rickettsial protein(s) required for ABM is uniformly localized to both poles of the bacterium and possibly throughout the rickettsial surface . Functional roles in rickettsial ABM for neuronal Wiskott-Aldrich syndrome protein (N-WASP) and the actin-related protein (Arp)2/3 complex, critical regulators of ABM of other pathogens, have not been established . Domains of N-WASP that have characterized inhibitory effects on N-WASP or Arp2/3 complex function were expressed in HeLa cells infected with R . rickettsii . Shigella flexneri-infected cells were used as a control . When ectopically expressed, the VCA domain of N-WASP (VCA) acts as a dominant/negative with respect to Arp2/3 complex function and N-WASP missing VCA (DeltaVCA) acts as a dominant/negative form of N-WASP . Expression of VCA or DeltaVCA severely inhibited S . flexneri ABM (no Shigella motility observed in the majority of expressing cells) while only moderately inhibiting ABM of R . rickettsii (approximately 35% decrease in the rate of ABM) . In addition, ectopically expressed full-length GFP-N-WASP was recruited by S . flexneri but not R . rickettsii, and Arp3 was detected by indirect immunofluorescence in S . flexneri actin tails but not within R . rickettsii actin tails . Collectively, these data suggest that rickettsial ABM is independent of N-WASP and Arp2/3 complex function.

Microb Pathog, 2003 Jul, 35(1), 11 - 8
The actin-based motility defect of a Shigella flexneri rmlD rough LPS mutant is not due to loss of IcsA polarity; Van den Bosch L et al.; Shigella flexneri requires the outer membrane protein IcsA(VirG) and lipopolysaccharide (LPS) for efficient actin-based motility (ABM) within mammalian cells which is essential for virulence . Wild type strains of S . flexneri 2a such as 2457T have smooth LPS whose O antigen (Oag) chains have two modal lengths and IcsA predominantly located at one pole on their cell surface . In contrast, rough LPS mutants lack Oag chains, have IcsA on lateral and polar regions of the cell surface, and are defective for ABM . In this study we directly compared the phenotype of a S . flexneri producing non-IcsP/SopA cleavable IcsA (IcsA*) with that of a rough LPS mutant . IcsA* was located on lateral and polar regions of smooth LPS bacteria, and was fully functional in ABM assays (HeLa cell monolayer plaque and F-actin comet tail formation) which contrasts with the R-LPS phenotype . This indicates that loss of polar IcsA localisation in R-LPS mutants is unrelated to their ABM defect, and suggests that Oag may directly contribute to IcsA-mediated ABM.

J Appl Microbiol, 2003, 95(2), 273 - 8
Intracellular vacuolation induced by culture filtrates of Plesiomonas shigelloides isolated from environmental sources; Falcon R et al.; AIMS: Potential virulence factors produced by culture filtrates of Plesiomonas shigelloides isolated from water were investigated . METHODS AND RESULTS: Culture filtrates of P . shigelloides strains were assayed for cytotoxic activity in CHO (Chinese hamster ovary), Vero (African green monkey kidney), HeLa (human cervix), HT29 (human epithelial intestinal) and SK6 (swine epithelial kidney) cells . Microscopic analyses revealed intensive cytoplasmic vacuolation including cell rounding and swelling, with gradual destruction of the monolayer in filtrate-treated cells . Neutral red assays showed that CHO, HeLa and Vero cells were the most sensitive to the vacuolating activity, which was evident within 30 min of culture filtrate exposure . This activity was inactived by heating at 56 degrees C for 15 min and partially neutralized by antiserum to the cytotoxin of Aeromonas hydrophila . All P . shigelloides strains had a cell-associated haemolysin in the agar plate assay . Three isolates were found to produce a cell-free haemolytic activity at 37 degrees C . In the suckling mouse test, two P . shigelloides culture supernatants were positive for enterotoxic activity . CONCLUSIONS: P . shigelloides culture filtrates isolated from aquatic environment cause intracellular vacuolation on mammalian cells, and produce haemolytic and enterotoxic activities . SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Plesiomonas strains.

Nat Cell Biol, 2003 Aug, 5(8), 720 - 6
Connexin-dependent inter-cellular communication increases invasion and dissemination of Shigella in epithelial cells; Tran Van Nhieu G et al.; Shigella flexneri, the causative agent of bacillar dystentery, invades the colonic mucosa where it elicits an intense inflammatory reaction responsible for destruction of the epithelium . During cell invasion, contact with host cells activates the type-III secretion of the Shigella IpaB and IpaC proteins . IpaB and IpaC are inserted into host cell plasma membranes and trigger initial signals that result in actin polymerization, while allowing cytosolic access of other bacterial effectors that further reorganize the cytoskeleton . After internalization, Shigella moves intracellularly and forms protrusions that infect neighbouring cells, promoting bacterial dissemination across the epithelium . Here, we show that during cell invasion, Shigella induces transient peaks in intracellular calcium concentration that are dependent on a functional type-III secretory apparatus . In addition, Shigella invasion induces the opening of Connexin 26 (Cx26) hemichannels in an actin- and phospholipase-C-dependent manner, allowing release of ATP into the medium . The released ATP, in turn, increases bacterial invasion and spreading, as well as calcium signalling induced by Shigella . These results provide evidence that pathogen-induced opening of connexin channels promotes signalling events that favour bacterial invasion and dissemination.

J Clin Microbiol, 2003 Jul, 41(7), 3078 - 88
Molecular epidemiology of Shigella in a Taiwan township during 1996 to 2000; Chen JH et al.; A previously identified Shigella flexneri serotype 2a strain was responsible for an outbreak of shigellosis in a Taiwan township in August 1996 . In order to find the relationship between this outbreak strain and subsequent Shigella infections in the area, 59, 47, 35, and 20 Shigella isolates recovered in 1997, 1998, 1999, and 2000, respectively, were collected and typed by serological and pulsed-field gel electrophoresis (PFGE) techniques . Of these 161 isolates, 139 isolates were S . flexneri serotype 2a, and one-third of them (47 isolates) exhibited the outbreak pattern . The remaining 92 S . flexneri serotype 2a isolates displayed 49 different NotI-PFGE patterns . Forty-five patterns were closely related to the outbreak pattern, with deletions of three specific NotI fragments occurring with high frequency . While the outbreak strain remained the main cause of shigellosis after the outbreak, the continuous emergence of closely related though poorly transmissible strains from the outbreak strain contributed to the observed annual decrease of shigellosis in the area.

Natl Med J India, 2003, 16 Suppl 2, 15 - 9
Progress in the prevention and control of diarrhoeal diseases since Independence; Bhattacharya SK; Acute diarrhoeal diseases constitute one of the major health problems among young children in India . It was estimated in 1978 that 1.5 million children under the age of 5 years die due to diarrhoea every year, which declined to 0.6-0.7 million in the estimate revised in 1992 . A similar declining trend has also been noted in hospitalized cases in Calcutta (present Kolkata) during 1980-95 as well as from other parts of India . Even today, cholera epidemics occur regularly in India . The cholera epidemic caused by a novel strain of Vibrio cholerae, designated as V . cholerae 0139 Bengal in 1992 and multidrug-resistant shigellosis in eastern India in 1984 are matters of grave concern . The launching of the National Diarrhoeal Diseases Control Programme (CDD) in 1978, based on a three-tier approach, is of great importance . The rate of use of oral rehydration salt (ORS) solution and oral rehydration therapy (ORT) remain suboptimal in India . In spite of the launching of the 'Ganga Action Plan' and the 'National River Action Plan', India faces a major problem of diarrhoeal diseases . Lack of safe water supply, poor environmental sanitation, improper disposal of human excreta and poor personal hygiene help to perpetuate and spread diarrhoeal diseases in India . Since diarrhoeal diseases are caused by 20-25 pathogens, vaccination, though an attractive disease prevention strategy, is not feasible . However, as the majority of childhood diarrhoeas are caused by V . cholerae, Shigellae dysenteriae type 1, rotavirus and enterotoxigenic Escherichia coli (E . coli) which have a high morbidity and mortality, vaccines against these organisms are essential for the control of epidemics . A strong political will with appropriate budgetary allocation is essential for the control of childhood diarrhoeal diseases in India, a formidable task in a country with a population of over 1 billion.

Cell Microbiol, 2003 Jul, 5(7), 481 - 91
A newborn mouse model for the study of intestinal pathogenesis of shigellosis; Fernandez MI et al.; Shigella infection is characterized by the induction of acute inflammation, which is responsible for the massive tissue destruction of the intestinal mucosa . A murine model would be a valuable tool for gaining a better understanding of the physiopathology of shigellosis and the host immune response to Shigella infection, but adult mice do not develop disease upon oral inoculation . We therefore attempted to develop a model of infection in newborn mice . Four-day-old mice inoculated with 50 microl of 5 x 10(9) invasive wild-type Shigella flexneri 5a were susceptible to bacterial infection, but mice inoculated with the non-invasive strain BS176 were not . Histologically, 4-day-old mice infected with the invasive strain presented intestinal lesions and inflammation similar to those described in patients with shigellosis . Moreover, cytokine and chemokine responses consistent with inflammation were observed . Lower bacterial inocula induced less severe intestinal damage . In contrast, 5-day-old mice inoculated with either the invasive or the non-invasive strain were not infected . We have thus established a mouse model that is suitable for the study of the pathogenesis of intestinal Shigella infection.

J Biol Chem, 2003 Sep 5, 278(36), 33878 - 86 Epub 2003 Jun 17.
The invasive phenotype of Shigella flexneri directs a distinct gene expression pattern in the human intestinal epithelial cell line Caco-2; Pedron T et al.; Invasion of the human colonic epithelium by Shigella flexneri causes inflammation that disrupts the intestinal barrier . Invaded intestinal epithelial cells are the major source of mediators recruiting the inflammatory infiltrate . To better characterize the global response of intestinal epithelial cells to Shigella invasion, Caco-2 cells were infected by an invasive isolate of S . flexneri 5a, and their transcriptome was analyzed by Affymetrix (Santa Clara, CA) microarrays (12,000 genes) and compared with these elicited by a non-invasive Shigella mutant and tumor necrosis factor (TNF)-alpha . The invasive and non-invasive strains enhanced transcription of a common pattern of 240 genes, among which genes encoding isoforms of cytochrome P-450 were induced . These genes were not induced by TNF-alpha . Conversely, both the invasive strain and TNF-alpha induced a common set of 18 genes, mainly encoding proinflammatory molecules . They also induced specific sets of genes . The transcriptome induced by the invasive strain was characterized by the induction of early genes (i.e . expressed within the first 45 min of invasion) and late genes (i.e . after 60 min of invasion) whose pattern was strongly biased toward stimulation of granulopoiesis, chemoattraction, activation, and adherence of polymorphonuclear leukocytes . When compared with a non-invasive Shigella and TNF-alpha, invasive Shigella induced a narrow transcriptome that seems to program infected epithelial cells to recruit a mucosal polymorphonuclear leukocyte to infiltrate . Dramatic increase in IL-8 gene transcription points to this chemokine as the major molecule orchestrating mucosal inflammation in shigellosis.

Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 875 - 83
Phylogenetic analysis of members of the genus Aeromonas based on gyrB gene sequences; Yanez MA et al.; The phylogenetic relationships of all known species of the genus Aeromonas were investigated by using the sequence of gyrB, a gene that encodes the B-subunit of DNA gyrase . Nucleotide sequences of gyrB were determined from 53 Aeromonas strains, including some new isolates, which were also characterized by analysis of the 16S rDNA variable regions . The results support the recognition of the family Aeromonadaceae, as distinct from Plesiomonas shigelloides and other enteric bacteria . This phylogenetic marker revealed strain groupings that are consistent with the taxonomic organization of all Aeromonas species described to date . In particular, gyrB results agreed with 16S rDNA analysis; moreover, the former showed a higher capacity to differentiate between species . The present analysis was useful for the elucidation of reported discrepancies between different DNA-DNA hybridization sets . Additionally, due to the sequence diversity found at the intraspecies level, gyrB is proposed as a useful target for simultaneous identification of species and strains . In conclusion, the gyrB gene has proved to be an excellent molecular chronometer for phylogenetic studies of the genus Aeromonas.

EMBO J, 2003 Jun 16, 22(12), 3050 - 61
A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity; Grosse R et al.; Vasodilator-stimulated phosphoprotein (VASP) is involved in multiple actin-mediated processes, including regulation of serum response factor (SRF) activity . We used the SRF transcriptional assay to define functional domains in VASP and to show that they coincide with those required for F-actin accumulation, as determined by a quantitative FACS assay . We identified inactive VASP mutants that can interfere both with F-actin assembly and with SRF activation by wild-type VASP . These VASP mutants also inhibit actin-based motility of Vaccinia virus and Shigella flexneri . VASP-induced F-actin accumulation and SRF activation require both functional Rho and its effector mDia, and conversely, mDia-mediated SRF activation is critically dependent on functional VASP . VASP and mDia also associate physically in vivo . These findings show that VASP and mDia function cooperatively downstream of Rho to control F-actin assembly and SRF activity.

FEBS Lett, 2003 Jun 19, 545(2-3), 183 - 7
In vitro DNA-binding properties of VirB, the Shigella flexneri virulence regulatory protein; McKenna S et al.; The DNA-binding activity of the Shigella flexneri VirB transcription factor was studied in vitro . The protein was found to bind non-specifically to DNA, but showed preferential binding to VirB-dependent promoter sequences . DNA binding was contingent on the presence of an intact helix-turn-helix motif . While high molecular mass protein-DNA complexes were formed in both specific and non-specific interactions with DNA, mutant derivatives of VirB lacking a leucine zipper domain or a carboxyl-terminal-located oligomerisation domain formed discrete complexes, indicating that an ability to oligomerise on DNA was responsible for the formation of high molecular mass complexes by the wild-type protein.

MMWR Morb Mortal Wkly Rep, 2003 Jun 6, 52(22), 513 - 6
Surveillance data from swimming pool inspections--selected states and counties, United States, May--September 2002; Antimicrobial and antioxidant activities of three Mentha species essential oils; University of Novi Sad, Faculty of Natural Sciences and Mathematics, Department of Chemistry, Novi Sad, FR Yugoslavia . mimica@ih.ns.ac.yu

The present study describes the antimicrobial activity and free radical scavenging capacity (RSC) of essential oils from Mentha aquatica L., Mentha longifolia L., and Mentha piperita L . The chemical profile of each essential oil was determined by GC-MS and TLC . All essential oils exhibited very strong antibacterial activity, in particularly against Esherichia coli strains . The most powerful was M . piperita essential oil, especially towards multiresistant strain of Shigella sonei and Micrococcus flavus ATTC 10,240 . All tested oils showed significant fungistatic and fungicidal activity {expressed as minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values, respectively}, that were considerably higher than those of the commercial fungicide bifonazole . The essential oils of M . piperita and M . longifolia were found to be more active than the essential oil of M . aquatica . Especially low MIC (4 microL/mL) and MFC (4 microL/mL) were found with M . piperita oil against Trichophyton tonsurans and Candida albicans (both 8 microL/mL) . The RSC was evaluated by measuring the scavenging activity of the essential oils on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and OH radicals . All examined essential oils were able to reduce DPPH radicals into the neutral DPPH-H form, and this activity was dose-dependent . However, only the M . piperita oil reduced DPPH to 50 % (IC50 = 2.53 microg/mL) . The M . piperita essential oil also exhibited the highest OH radical scavenging activity, reducing OH radical generation in the Fenton reaction by 24 % (pure oil) . According to GC-MS and TLC (dot-blot techniques), the most powerful scavenging compounds were monoterpene ketones (menthone and isomenthone) in the essential oils of M . longifolia and M . piperita and 1,8-cineole in the oil of M . aquatica.

Folia Microbiol (Praha), 2003, 48(2), 139 - 47
The Escherichia fergusonii iucABCD iutA genes are located within a larger chromosomal region similar to pathogenicity Islands; Smajs D et al.; Three strains of Escherichia fergusonii (EF873, EF1496, EF939) of 50 strains tested produced the hydroxamate siderophore aerobactin . Screening of a cosmid library of the strain EF873 chromosomal DNA (in aerobactin nonproducing Escherichia coli VCS257) for aerobactin production identified iucABCD and iutA gene orthologues . The predicted IucABCD and IutA proteins showed 59-65% identity to the corresponding proteins of Shigella flexneri and E . coli . Aerobactin molecules synthesized by E . fergusonii and E . coli strains stimulated growth of aerobactin indicator strains harboring either E . coli or E . fergusonii iutA genes . In the 12 kb upstream and 17 kb downstream regions of the iuc and iut genes, 20 additional ORFs were identified . Their gene products showed homology to proteins from E . coli, S . flexneri, Klebsiella aerogenes, Pseudomonas aeruginosa and Vibrio cholerae . Probes recognizing DNA sequences from a region of more than 25 kb, which included the iucABCD and iutA genes, hybridized with chromosomal DNA of two aerobactin-producing strains (EF873 and EF939), but not with other nonproducing E . fergusonii strains tested . These data, together with the genetic organization of this region, suggest that E . fergusonii iucABCD iutA genes are a portion of a larger segment of DNA similar to pathogenicity islands of other bacteria.

World J Gastroenterol, 2003 Jun, 9(6), 1347 - 51
Identification of RanBMP interacting with Shigella flexneri IpaC invasin by two-hybrid system of yeast; Yao X et al.; AIM: Bacillary dysentery caused by Shigella flexneri is still a threat to human health . Of four invasion plasmid antigen proteins (IpaA,B,C and D), IpaC plays an important role in the pathogenicity of this pathogen . The purpose of this study was to investigate the proteins interacting with IpaC in the host cell during the pathogenic process of this disease . METHODS: By applying two-hybrid system, the bait plasmid containing ipaC gene was constructed and designated pGBKT-ipaC . The bait plasmid was transformed AH109, and proved to express IpaC and then HeLa cDNA library plasmids were introduced into the above transformed AH109 . The transformation mixture was plated on medium lacking Trp, Leu, and His in the initial screen, then restreaked on medium lacking Trp, Leu, His and Ade . Colonies growing on the selection medium were further assayed for beta-galactosidase activity . BLAST was carried out in the database after sequencing the inserted cDNA of the positive library plasmid . RESULTS: Among the 2X10(6) transformants, 64 positive clones were obtained as determined by activation of His, Ade and LacZ reporter genes . Sequence analysis revealed that cDNA inserts of two colonies were highly homologous to a known human protein, RanBPM . CONCLUSION: These results provide evidence that IpaC may be involved in the invasion process of S . flexneri by interacting with RanBPM, and RanBPM is most likely to be the downstream target of IpaC in the cascade events of S . flexneri infection.

Vaccine, 2003 Jun 20, 21(21-22), 3043 - 50
Protective efficacy of oral immunization with heat-killed Shigella flexneri 2a in animal model: study of cross protection, immune response and antigenic recognition; Mukhopadhaya A et al.; Oral immunization of rabbits with four doses of 10(11) heat-killed Shigella flexneri 2a showed 100% protection against challenge with virulent S . flexneri 2a . After orally immunizing Guinea pigs with four doses of heat-killed S . flexneri 2a 100% protection could be shown against ocular challenge with the same virulent S . flexneri 2a strain but this conferred no protection against challenge with Shigella dysenteriae type 1 . In enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments both whole cell lysate-envelope (WCL-E) fraction and outer membrane proteins (OMPs) were recognized by the antisera . Though protective mechanism in shigellosis is not established with certainty, outer membrane proteins (specially 38, 34, 23 and 20kDa proteins) may be the major antigens in the induction of protective immune responses as indicated by this observation.

Clin Anat, 2003 Jul, 16(4), 317 - 27
Vascular changes in duodenal mucosa in shigellosis and cholera; Koshi R et al.; Vascular endothelial cells are highly specialized cells with numerous sensory and modulator functions . Our previous studies show extensive microvascular changes in rectal mucosal vasculature of patients with acute infective diarrhea (Mathan and Mathan 1985a, Gut 26:710-717).We looked for changes in the duodenal mucosal vasculature in two naturally occurring diarrheal infections: shigellosis and cholera . Duodenal mucosal biopsies from 14 patients with shigellosis, 12 patients with cholera, and 10 healthy volunteers were examined under the electron microscope . There were extensive microvascular changes in the duodenum in shigellosis and cholera . Congestion and dilatation of capillaries and venules, stagnation of blood, thinning of the endothelial lining, and platelet clumping were commonly seen in both conditions . Endothelial damage was also common to both conditions but was mild to moderate in cholera and severe in shigellosis with frank hemorrhage, frequent formation of stress fibers, widening of intercellular spaces, cytoplasmic blebbing, cell fragmentation, and intravascular thrombosis . Erythrocyte aggregates, platelet aggregates, and leucocyte plugging lead to capillary obstruction . The arterioles were severely constricted . These changes in the endothelial lining of the microvasculature could contribute to the pathogenesis of the disease resulting in peripheral vascular insufficiency, inadequate oxygen delivery to intestine, and organ dysfunction . The factors influencing these changes, their implications, and possible therapeutic interventions are discussed .

Microbes Infect, 2003 Jun, 5(7), 579 - 92
Prevalence and molecular diversity of pHS-2 plasmids, marker for arthritogenicity, among clinical Escherichia coli Shigella isolates; Adam T et al.; Reactive arthritis can occur after numerous bacterial infections, including bacillary dysentery caused by Escherichia coli Shigella . A major risk factor for the disease is the HLA B27 phenotype in the human host . By comparison between plasmid profiles of arthritogenic vs . nonarthritogenic Shigella strains, the pHS-2 plasmid has been previously associated with the arthritogenic capacity of Shigella isolates . However, the prevalence of this plasmid in the various Shigella biotypes and serotypes is largely unknown . On this background, 188 clinical isolates from intestinal disease representing all 46 Shigella serogroups were studied for the presence of the pHS-2 plasmid, using PCR, dot blot and Southern blot techniques and by analysis of restriction fragment length polymorphisms . The pHS-2 plasmid was found in nine of 14 E . coli Flexneri serogroups, in E . coli Dysenteriae 1 and in E . coli Boydii 16 . In addition, we show marked variability of this plasmid in E . coli Flexneri 3A and 4A strains . Major biological diversity of the pHS-2 plasmid was found to be strictly related to Shigella serogroups . The prevalence pattern of the pHS-2 plasmid matches published data on arthritogenic Shigella isolates, providing additional indirect evidence for the potential validity of this plasmid as a marker for arthritogenicity.

Mol Microbiol, 2003 Jun, 48(5), 1401 - 16
Three-way interactions among the Sfh, StpA and H-NS nucleoid-structuring proteins of Shigella flexneri 2a strain 2457T; Deighan P et al.; Shigella flexneri 2a strain 2457T has been found to express Sfh, a new member of the H-NS-like family of nucleoid-structuring proteins . With H-NS and its paralogue, StpA, this brings to three the number of these proteins expressed in this bacterium . This raises the possibility that three-way interactions may occur in S . flexneri among these proteins and between the proteins and each other's genes . Such three-way interactions among H-NS-like proteins have not been described previously . The expression of the sfh, stpA and hns genes was studied at the transcriptional and post-transcriptional levels . The Sfh protein displays growth phase-dependent regulation that distinguishes it from both H-NS and StpA . Like H-NS and StpA, Sfh can bind to its own promoter region, it negatively autoregulates transcription of its own gene, and when overexpressed all three proteins cross-repress transcription of each other's genes . The presence of highly conserved oligomerization domains within these molecules suggested the possibility of protein-protein interactions . Like H-NS and StpA, the purified Sfh protein forms homodimers in solution . Using the yeast two-hybrid assay we show that each of the three proteins also forms homodimers in vivo and, additionally, each protein can form heterodimers with either of its homologues . This raises the possibility that Sfh may modulate the activities of H-NS and StpA, and vice versa.

APMIS, 2003 Apr, 111(4), 477 - 82
Effects of Ocimum gratissimum L essential oil at subinhibitory concentrations on virulent and multidrug-resistant Shigella strains from Lagos, Nigeria; Iwalokun BA et al.; Ocimum gratissimum leaf extracts have been extensively demonstrated to be effective against the various aetiologic agents of diarrhoea, including Shigellae . However, the mechanism of the shigellocidal action of this plant remains to be understood . This study investigated the effects of O . gratissimum essential oil (EO) at subinhibitory concentrations of 0.75 and 1.0 microg/ml on virulence and multidrug-resistant strains of 22 Shigella isolates from Nigeria . Compared with untreated Shigella strains, O . gratissimum EO caused significant decreases (p<0.01) in extracellular protease activity, o-lipopolysaccharide rhamnose content and incidence of invasiveness mediated as keratoconjunctivitis in guinea pig . The disparity in extracellular protease activity and o-lipopolysacharide rhamnose between the two treatment groups was also found to be significant (p<0.05), suggesting greater anti-virulent effects of O . gratissimum oil at 1.0 microg/ml . Antibiotic susceptibility testing revealed that the EO of O . gratissimum reduced the MICs of antibiotics to which Shigellae showed resistance by 9.8-53.1% and fluoroquinolones by 18.2-45.5% . The results of this study strongly suggest inhibition of extracellular protease and expression of O-LPS rhamnose in Shigellae by O . gratissimum EO . The future use of O . gratissimum- antibiotic combinations as a therapeutic measure against shigellosis is discussed.

FEMS Microbiol Lett, 2003 May 28, 222(2), 289 - 95
The IS1 elements in Shigella boydii: horizontal transfer, vertical inactivation and target duplication; Hsu WB et al.; IS1(SB) and its two variants were identified as the major and minor IS1 elements in Shigella boydii . The nucleotide sequences of IS1(SB), IS1(O157:H7) from Escherichia coli O157:H7 and IS1F from E . coli K12 suggest that these IS1 elements had been horizontally transferred among S . boydii and E . coli O157:H7 and K12 . The two IS1(SB) variants and IS1(O157:H7) have transposition activities 7- to 86-fold less than that of IS1(SB), whereas IS1F has little transposition activity . Analysis of the flanking sequences of IS1(SB) and its two variants in S . boydii revealed the nature of regional specificity of the target sites and the sequence dependence of 8 and 9 bp target duplications, for which a model is presented.

FEMS Microbiol Lett, 2003 May 28, 222(2), 273 - 80
A method for allelic replacement in Francisella tularensis; Golovliov I et al.; A vector for mutagenesis of Francisella tularensis was constructed based on the pUC19 plasmid . By inserting the sacB gene of Bacillus subtilis, oriT of plasmid RP4, and a chloramphenicol resistance gene of Shigella flexneri, a vector, pPV, was obtained that allowed specific mutagenesis . A protocol was developed that allowed introduction of the vector into the live vaccine strain, LVS, of F . tularensis by conjugation . As a proof of principle, we aimed to develop a specific mutant defective in expression of a 23-kDa protein (iglC) that we previously have shown to be prominently upregulated during intracellular growth of F . tularensis . A plasmid designated pPV-DeltaiglC was developed that contained only the regions flanking the encoding gene, iglC . By a double crossover event, the chromosomal iglC gene was deleted . However, the resulting strain, denoted DeltaiglC1, still had an intact iglC gene . Southern blot analysis verified that LVS harbors two copies for the iglC gene . The mutagenesis was therefore repeated and a mutant defective in both iglC alleles, designated DeltaiglC1+2, was obtained . The DeltaiglC1+2 strain, in contrast to DeltaiglC1, was shown to display impaired intracellular macrophage growth and to be attenuated for virulence in mice . The developed genetic system has the potential to provide a tool to elucidate virulence mechanisms of F . tularensis and the specific F . tularensis mutant illustrates the critical role of the 23-kDa protein, iglC, for the virulence of F . tularensis LVS.

J Insect Physiol, 2002 Jun, 48(6), 601 - 608
Adipokinetic hormone enhances laminarin and bacterial lipopolysaccharide-induced activation of the prophenoloxidase cascade in the African migratory locust, Locusta migratoria; Goldsworthy G et al.; Lom-AKH-I enhances the activation in vivo of prophenoloxidase in the haemolymph of the African migratory locust, Locusta migratoria, in response to challenge with laminarin . AKH does not influence the speed or initial magnitude of the phenoloxidase response to laminarin, but prolongs the period of activation of the enzyme in a dose-dependent manner . Injections of preparations of bacterial lipopolysaccharide (LPS) do not activate prophenoloxidase in vivo, but co-injection of Lom-AKH-I with commercial preparations of LPS from Klebsiella pneumoniae, Escherichia coli, or Shigella flexneri (but not one from Pseudomonas aeroginosa) results in dose-dependent increases in the levels of phenoloxidase that persist in the haemolymph for several hours . It is argued that the effects of AKH on phenoloxidase activation in locusts described here are, at least in part, related directly to changes in lipid metabolism brought about by the hormone.

Int J Med Microbiol, 2003 Apr, 293(1), 55 - 67
Shigella interaction with intestinal epithelial cells determines the innate immune response in shigellosis; Fernandez MI et al.; Shigellae are Gram-negative bacilli that cause bacillary dysentery in humans . This review summarizes current knowledge of Shigella pathogenesis and pathogenicity factors, invasion of epithelial cells, intracellular motility and cell-to-cell spreading, as well as components of the host cell involved in innate immune responses.

Saudi Med J, 2003 Apr, 24(4), 403 - 5
Shigella flexneri bacteremia in a child; Bello CS et al.; We report an unusual and lethal case of Shigella flexneri septicemia in an 8-year-old Saudi handicapped child from a social home presenting with severe toxic megacolon and acute abdomen secondary to fulminant necrotizing enterocolitis.

Mol Microbiol, 2003 May, 48(4), 913 - 31
IcsB, secreted via the type III secretion system, is chaperoned by IpgA and required at the post-invasion stage of Shigella pathogenicity; Ogawa M et al.; Shigella deliver a subset of effector proteins such as IpaA, IpaB and IpaC via the type III secretion system (TTSS) into host cells during the infection of colonic epithelial cells . Many bacterial effectors including some from Shigella require specific chaperones for protection from degradation and targeting to the TTSS . In this study, we have investigated the role of the icsB gene located upstream of the ipaBCDA operon in Shigella infection because the role of IcsB as a virulence factor remains unknown . Here, we found that the IcsB protein is secreted via the TTSS of Shigella in vitro and in vivo . We show that IpgA protein encoded by ipgA, the gene immediately downstream of icsB, serves as the chaperone required for the stabilization and secretion of IcsB . We have shown that IcsB binds to IpgA in bacterial cytosol and the binding site is in the middle of the IcsB protein . Intriguingly, although its significance in Shigella pathogenicity is as yet unclear, the icsB gene can be read-through into the ipgA gene to create a translational fusion protein . Furthermore, the contribution of IcsB to the pathogenicity of Shigella was demonstrated by plaque-forming assay and the Sereny test . The ability of the icsB mutant to form plaques was greatly reduced compared with that of the wild type in MDCK cell monolayers . Furthermore, when guinea pig eyes were infected with a non-polar icsB mutant, the bacteria failed to provoke keratoconjunctivitis . These results suggest that IcsB is secreted via the TTSS, chaperoned by IpgA, and required at the post-invasion stage of Shigella pathogenicity

FEMS Immunol Med Microbiol, 2003 May 25, 36(3), 117 - 26
Helicobacter pylori: an invading microorganism? A review; Petersen AM et al.; In this review we evaluate the pros and cons of Helicobacter pylori invasion of epithelial cells as part of the natural history of H . pylori infection . H . pylori is generally considered an extracellular microorganism . However, a growing body of evidence supports the controversial hypothesis that at least a subset of H . pylori microorganisms has an intracellular (intraepithelial) location . Most significant is the fact that H . pylori invades cultured epithelial cells with invasion frequencies similar to Yersinia enterocolitica and better than Shigella flexneri; furthermore, studies of invasion mechanisms suggest that H . pylori invasion of and survival within epithelial cells is not merely a passive event, but requires active participation of the microorganism . Although many studies of human gastric biopsy specimens have failed to demonstrate any intracellular H . pylori, some studies have revealed a minor fraction of H . pylori inside gastric epithelial cells, with possible linkage to peptic ulceration and epithelial cell damage . In conclusion, these data encourage further research to establish whether intracellular H . pylori does play a role in H . pylori colonization of the human stomach and in peptic ulcer pathogenesis.

J Clin Microbiol, 2003 May, 41(5), 1919 - 24
Antimicrobial resistance and genetic diversity of Shigella sonnei isolates from western Ireland, an area of low incidence of infection; DeLappe N et al.; Shigella sonnei is a significant cause of gastroenteritis in both developing and industrialized countries . Definition of the diversity and antimicrobial susceptibility of S . sonnei isolates may be helpful in the management of individual cases and outbreaks . Antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) were performed with 67 isolates of S . sonnei predominantly (n = 59) from three counties in the west of Ireland . Phage typing (n = 17), plasmid profiling (n = 28), and integron analysis (n = 24) were performed with subsets of strains . PFGE typing permitted recognition of two major clusters: PFGE type A (n = 53) and PFGE type B (n = 14) . PFGE type A was associated with resistance to ampicillin, streptomycin, and sulfonamides (51 of 53 isolates), and those that were phage typed (n = 6) were phage type 3 . PFGE type B was associated with resistance to streptomycin, sulfonamides, tetracycline, and trimethoprim (11 of 14 isolates) and phage type 6 (9 of 11 isolates) . Fifteen different plasmid profiles were identified among the 28 isolates analyzed . A class 2 integron was present in all 14 PFGE type B isolates . One of these isolates also contained a class 1 integron and showed a unique variant of the PFGE type B pattern . Sequence analysis of the gene cassette structures contained within these integrons identified distinct open reading frames that encoded determinants of resistance to trimethoprim, streptomycin, and streptothricin . Our data demonstrate two predominant PFGE types among S . sonnei isolates circulating in this region . The limited diversity of the S . sonnei isolates in this region means that detection of isolates indistinguishable by PFGE and according to their antibiograms in two or more patients is not persuasive evidence of a common-source food- or waterborne outbreak . Indistinguishable plasmid profiles in addition to indistinguishable PFGE and antibiogram types may be more suggestive of an epidemiologically relevant link between cases.

J Biol Chem, 2003 Jul 11, 278(28), 26086 - 93 Epub 2003 May 05.
Sequential interaction of actin-related proteins 2 and 3 (Arp2/3) complex with neural Wiscott-Aldrich syndrome protein (N-WASP) and cortactin during branched actin filament network formation; Uruno T et al.; The WASP and cortactin families constitute two distinct classes of Arp2/3 modulators in mammalian cells . Physical and functional interactions among the Arp2/3 complex, VCA (a functional domain of N-WASP), and cortactin were examined under conditions that were with or without actin polymerization . In the absence of actin, cortactin binds significantly weaker to the Arp2/3 complex than VCA . At concentrations of VCA 20-fold lower than cortactin, the association of cortactin with the Arp2/3 complex was nearly abolished . Analysis of the cells infected with Shigella demonstrated that N-WASP located at the tip of the bacterium, whereas cortactin accumulated in the comet tail . Interestingly, cortactin promotes Arp2/3 complex-mediated actin polymerization and actin branching in the presence of VCA at a saturating concentration, and cortactin acquired 20 nm affinity for the Arp2/3 complex during actin polymerization . The interaction of VCA with the Arp2/3 complex was reduced in the presence of both cortactin and actin . Moreover, VCA reduced its affinity for Arp2/3 complex at branching sites that were stabilized by phalloidin . These data imply a novel mechanism for the de novo assembly of a branched actin network that involves a coordinated sequential interaction of N-WASP and cortactin with the Arp2/3 complex.

Lancet Infect Dis, 2003 May, 3(5), 275 - 81
Effect of washing hands with soap on diarrhoea risk in the community: a systematic review; Curtis V et al.; We set out to determine the impact of washing hands with soap on the risk of diarrhoeal diseases in the community with a systematic review with random effects meta-analysis . Our data sources were studies linking handwashing with diarrhoeal diseases . Seven intervention studies, six case-control, two cross-sectional, and two cohort studies were located from electronic databases, hand searching, and the authors' collections . The pooled relative risk of diarrhoeal disease associated with not washing hands from the intervention trials was 1.88 (95% CI 1.31-2.68), implying that handwashing could reduce diarrhoea risk by 47% . When all studies, when only those of high quality, and when only those studies specifically mentioning soap were pooled, risk reduction ranged from 42-44% . The risks of severe intestinal infections and of shigellosis were associated with reductions of 48% and 59%, respectively . In the absence of adequate mortality studies, we extrapolate the potential number of diarrhoea deaths that could be averted by handwashing at about a million (1.1 million, lower estimate 0.5 million, upper estimate 1.4 million) . Results may be affected by the poor quality of many of the studies and may be inflated by publication bias . On current evidence, washing hands with soap can reduce the risk of diarrhoeal diseases by 42-47% and interventions to promote handwashing might save a million lives . More and better-designed trials are needed to measure the impact of washing hands on diarrhoea and acute respiratory infections in developing countries.

FEMS Microbiol Lett, 2003 Apr 25, 221(2), 263 - 7
Subunit interaction of monomeric alanine racemases from four Shigella species in catalytic reaction; Yokoigawa K et al.; Bacterial alanine racemases are classified into two types of subunit structure (monomer and homodimer) . To clarify the catalytic unit of monomeric alanine racemases, we examined the apparent molecular mass of the monomeric alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei by gel filtration in the presence of the substrate and inhibitor . The enzymes were eluted on gel filtration as a monomer of about 39,000 Da at low protein concentration and in the absence of L-alanine and D-cycloserine . An increase in the apparent molecular mass was induced by increasing the protein concentration or by adding the ligands in the elution buffer . The increase ratio depended on the ligand concentration, and the maximum apparent molecular masses of all enzymes were 60,000 and 76,000 Da in the presence of 100 mM L-alanine and 5 mM D-cycloserine, respectively . D-cycloserine may induce an inactive dimer and L-alanine may induce an intermediate between the monomer and dimer because of dynamic equilibrium . The apoenzyme also showed similar behavior in the presence of the ligands, but the increase ratios were lower than those of the holoenzymes . The Bacillus psychrosaccharolyticus alanine racemase, having a dimeric structure, showed a constant molecular mass irrespective of the absence or presence of the ligands . These results suggest that the monomeric Shigella Alr enzymes have a dimeric structure in the catalytic reaction . Substances that inhibit the subunit interaction of monomeric alanine racemases may be useful as a new type of antibacterial.

FEMS Microbiol Lett, 2003 Apr 25, 221(2), 213 - 9
Multicopy icsA is able to suppress the virulence defect caused by the wzz(SF) mutation in Shigella flexneri; Morona R et al.; The lipopolysaccharides (LPS) of Shigella flexneri are important for virulence and their O antigen (Oag) polysaccharide chains affect IcsA (VirG)-mediated actin-based motility (ABM) within mammalian cells . S . flexneri 2a 2457T has smooth LPS whose Oag chains have two modal lengths (short (S)-type and very long (VL)-type), and has IcsA predominantly located at one pole on its cell surface . A S . flexneri 2457T wzz(SF) mutant (RMA696) has VL-type Oag but not S-type Oag chains, less IcsA detectable by immunofluorescence on its cell surface, reduced virulence and defective ABM . Introduction of a plasmid encoding IcsA into S . flexneri wzz(SF) showed that multicopy icsA could suppress the virulence defects (Sereny reaction, HeLa cell monolayer plaquing, and F-actin comet tail formation) caused by the wzz(SF) mutation suggesting that the VL-type Oag chains were masking IcsA and limiting the amount available to initiate ABM.

FEMS Microbiol Lett, 2003 Apr 25, 221(2), 173 - 80
Lipopolysaccharide O antigen chains mask IcsA (VirG) in Shigella flexneri; Morona R et al.; Shigella flexneri 2a strain 2457T lipopolysaccharide (LPS) has O antigen (Oag) chains with two modal lengths (S-type and VL-type), and has IcsA apparently located at one pole on its cell surface . Treatment of Y serotype derivatives of 2457T and RMA696 (2457T wzz(SF)) with Sf6 tailspike protein (TSP) resulted in hydrolysis of Oag chains, and an increase in detection of IcsA by indirect immunofluorescence staining on both the lateral and polar regions of the cell surface . Newly synthesised IcsA expressed from a pBAD promoter in a S . flexneri Y strain was also detected on both the lateral and polar regions of the cell when incubated with TSP prior to immunofluorescence staining . We conclude that IcsA is actually located on both lateral and polar regions of the S . flexneri cell surface, and that LPS Oag chains mask the presence of IcsA by hindering its detection with antibodies . These results have implications for the mechanism of IcsA export . They suggest that while IcsA export is predominantly targeted to the old cell pole, it can also occur on the lateral regions of the cell surface.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Jul-Aug, (4 Suppl), 76 - 7
{Role of Shigella flexneri type-specific antigen in bacterial resistance to oxidative stressor}; Mal'shakova IL; Lac+ type- S . flexneri 1298 1a and S . flexneri 1338 1b hybrids has been obtained in the crosses of Shigella strains with Escherichia coli K-12 Hfr C . The detection of in vitro survive of the bacteria of genetically linked pair strains, differing in type specific antigen synthesis is presented . The more sensitive of lac+ type- hybrids to the treatment by hydrogen peroxide of concentration 205 mM in comparison with the parent S . flexneri strains possesses the type specific antigen synthesis was shown.

Virology, 2003 Mar 30, 308(1), 114 - 27
Morphology of temperate bacteriophage SfV and characterisation of the DNA packaging and capsid genes: the structural genes evolved from two different phage families; Allison GE et al.; The entire genome of SfV, a temperate serotype-converting bacteriophage of Shigella flexneri, has recently been sequenced (Allison, G.E., Angeles, D., Tran-Dinh, N., Verma, N.K . 2002, J . Bacteriol . 184, 1974-1987) . Based on the sequence analysis, we further characterised the SfV virion structure and morphogenesis . Electron microscopy indicated that SfV belongs to the Myoviridae morphology family . Analysis of the proteins encoded by orf1, orf2, and orf3 revealed that they were homologous to small and large terminase subunits, and portal proteins, respectively; the protein encoded by orf5 showed homology to capsid proteins . Western immunoblot of the phage with anti-SfV sera revealed two antigenic proteins, and the N-terminal amino acid sequence of the 32-kDa protein corresponded to amino acids 116 to 125 of the ORF5 protein, suggesting that the capsid may be processed . Functional analysis of orf4 showed that it encodes the phage capsid protease . The proteins encoded by orfs1, 2, 3, 4, and 5 are homologous to similar proteins in the Siphoviridae phage family of both gram-positive and gram-negative origin . The capsid and morphogenesis genes are upstream and adjacent to the genes encoding Myoviridae (Mu-like) tail proteins . The organisation of the structural genes of SfV is therefore unique as the head and tail genes originate from different morphology groups.

Infect Immun, 2003 May, 71(5), 2775 - 86
Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T; Wei J et al.; We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp) . Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli . The genome exhibits the backbone and island mosaic structure of E . coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E . coli . The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S . flexneri-specific genes . The 2457T genome was also compared with that of a recently sequenced S . flexneri 2a strain, 301 . Our data are consistent with Shigella being phylogenetically indistinguishable from E . coli . The S . flexneri-specific regions contain many genes that could encode proteins with roles in virulence . Analysis of these will reveal the genetic basis for aspects of this pathogenic organism's distinctive lifestyle that have yet to be explained.

Infect Immun, 2003 May, 71(5), 2684 - 92
Persistence of mucosal mast cells and eosinophils in Shigella-infected children; Raqib R et al.; Cells of the innate immune system and their mediators were studied at the single-cell level in the rectums of pediatric and adult patients with Shigella infection to better understand why children are at higher risk for severe infection . Adult patients had increased infiltration of mucosal mast cells (MMC) at the acute stage (3 to 5 days after the onset of diarrhea) and eosinophils in early convalescence (14 to 16 days after onset) . Increased expression of stem cell factor and prostaglandin H synthase-1 (PGHS-1) was associated with increased tryptase-K(i)67-double-positive MMC in the acute stage and increased apoptosis of MMC, which led to a rapid decline in early convalescence . The eosinophils demonstrated increased expression of major basic protein (MBP), eotaxin, and CCR3, as well as increased necrotic death . The neutrophils showed enhanced alpha-defensin and lactoferrin expression in the acute phase . In contrast to adults, the pediatric patients demonstrated delayed accumulation of mast cells and eosinophils, while alpha-defensin expression persisted during convalescence . In contrast, neutrophil counts and lactoferrin expression were reduced in children compared to adults . The results suggest that children with shigellosis have a persistent activation of the innate immune response in the convalescent phase, indicating delayed elimination of Shigella antigens compared to adults.

Nature, 2003 Apr 17, 422(6933), 775 - 81
Microbial pathogenesis and cytoskeletal function; Gruenheid S et al.; Pathogenic microbes subvert normal host-cell processes to create a specialized niche, which enhances their survival . A common and recurring target of pathogens is the host cell's cytoskeleton, which is utilized by these microbes for purposes that include attachment, entry into cells, movement within and between cells, vacuole formation and remodelling, and avoidance of phagocytosis . Our increased understanding of these processes in recent years has not only contributed to a greater comprehension of the molecular causes of infectious diseases, but has also revealed fundamental insights into normal functions of the cytoskeleton . From the use of bacterial toxins to investigate Rho family GTPases to in vitro studies of actin polymerization using Listeria and Shigella, the study of pathogenesis has provided important tools to probe cytoskeletal function.

J Food Prot, 2003 Apr, 66(4), 668 - 73
Antimicrobial effect of spices and herbs on Shigella sonnei and Shigella flexneri; Bagamboula CF et al.; Of 17 spices and herbs tested at 1% (wt/vol) in Mueller-Hinton (MH) agar, only cloves, thyme, oregano, allspice, basil, rosemary, and marjoram showed antimicrobial effects on Shigella . The MICs of thyme, oregano, basil, and rosemary (as determined by the agar dilution method) ranged from 0.5 to 1% (wt/vol) depending on the Shigella strain used . With the use of various combinations of temperatures (12, 22, and 37 degrees C), pHs (5.0, 5.5, and 6.0), and NaCl concentrations (1, 2, 3, and 4%, wt/vol) and the inclusion or exclusion of thyme or basil at 1% (wt/vol) in an MH agar model system, it was established that basil or thyme can contribute to combination processing as a growth-inhibitory factor for Shigella spp . In the presence of basil and thyme, Shigella flexneri did not develop CFU during the 7-day incubation period for, respectively, 14 and 16 of the 18 tested combinations, while growth was noted in the corresponding temperature-pH-NaCl concentration combinations without basil or thyme . A growth-inhibitory effect on Shigella sonnei was also noted . The results of an orientation study involving the addition of basil and thyme to spaghetti sauce prior to autoclaving and S . sonnei inoculation indicated that basil and thyme contributed to the reduction of S . sonnei after 16 days at 12 degrees C but not at 4 degrees C.

J Food Prot, 2003 Apr, 66(4), 535 - 41
Concurrent outbreaks of Shigella sonnei and enterotoxigenic Escherichia coli infections associated with parsley: implications for surveillance and control of foodborne illness; Naimi TS et al.; In recent years, the globalization of the food supply and the development of extensive food distribution networks have increased the risk of foodborne disease outbreaks involving multiple states or countries . In particular, outbreaks associated with fresh produce have emerged as an important public health concern . During July and August 1998, eight restaurant-associated outbreaks of shigellosis caused by a common strain of Shigella sonnei occurred in the United States and Canada . The outbreak strain was characterized by unique pulsed-field gel electrophoresis patterns . Epidemiologic investigation determined that the illness was associated with the ingestion of parsley at four restaurants; at the other four restaurants, the majority of the people who contracted the illness ate parsley . Isolates from patrons in two unrelated restaurant-associated enterotoxigenic Escherichia coli (ETEC) outbreaks in Minnesota shared a common serotype and pulsed-field gel electrophoresis (PFGE) pattern . Parsley was the implicated or suspected source of both ETEC outbreaks . In each of the outbreak-associated restaurants, parsley was chopped, held at room temperature, and used as an ingredient or garnish for multiple dishes . Infected food workers at several restaurants may also have contributed to the propagation of the outbreak . The sources of parsley served in outbreak-associated restaurants were traced, and a 1,600-acre farm in Baja California, Mexico, was identified as a likely source of the parsley implicated in six of the seven Shigella outbreaks and as a possible source of the parsley implicated in the two ETEC outbreaks . Global food supplies and large distribution networks demand strengthened laboratory and epidemiologic capacity to enable state and local public health agencies to conduct foodborne disease surveillance and to promote effective responses to multistate outbreaks.

Infect Immun, 2003 Apr, 71(4), 2288 - 91
Bidirectional concentration-dependent effects of tumor necrosis factor alpha in Shigella dysenteriae-related seizures; Yuhas Y et al.; We have previously demonstrated that pretreatment of mice with Shigella dysenteriae sonicate enhanced their susceptibility to pentylenetetrazole-induced seizures and that tumor necrosis factor alpha (TNF-alpha) was proconvulsive in this respect . The present study shows that TNF-alpha, at high concentrations, may also exert a suppressive effect on Shigella-mediated seizures . This implies that high levels of TNF-alpha may play a protective role in neurologic complications of S . dysenteriae infection.

Di Yi Jun Yi Da Xue Xue Bao, 2003 Mar, 23(3), 230 - 2
Expression and purification of invasion plasmid antigen C of Shigella flexneri; Sun SX et al.; OBJECTIVE: To induce the expression of and purify invasion plasmid antigen C (IpaC) of Shigella flexneri for studying the pathogenesis of Shigella flexneri . METHODS: Prokaryotic expression plasmid pET32a-ipaC was constructed and incorporated into E.coli BL21 (lambda DE3) . The engineered bacteria were induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) to express IpaC, which was identified by SDS-PAGE and purified by QIA expressionist system . RESULTS: SDS-PAGE presented a band for the fusion protein with the relative molecular mass of approximately 63 000, whose expression reached up to 11% of the total protein of E.coli BL21(lambda DE3) . After proper purification, a purity of the target fusion protein of over 90% was achieved when the concentration of imidazole for elution was 350 mmol/L . CONCLUSION: The recombinant plasmid pET32a-ipaC has been stably and efficiently expressed in E.coli BL21 (lambda DE3), and QIA expressionist purification system proves to be simple and highly efficient.

Trends Microbiol, 2003 Mar, 11(3), 139 - 43
Exploiting host microtubule dynamics: a new aspect of bacterial invasion; Yoshida S et al.; During infection, many pathogenic bacteria modulate the actin cytoskeleton of eukaryotic host cells to facilitate various infectious processes such as the attachment to or invasion of epithelial cells . Additionally, some pathogenic bacteria are capable of modulating the dynamics of host microtubule (MTs) . Although the molecular basis for this is still poorly understood, a recent study of the Shigella VirA effector protein, which is delivered via a type III secretion system, suggests that MT destabilization plays an important role in Shigella infection.

Carbohydr Res, 2003 Mar 28, 338(7), 589 - 96
Synthesis of the pentasaccharide related to the repeating unit of the antigen from Shigella dysenteriae type 4 in the form of its methyl ester 2-(trimethylsilyl)ethyl glycoside; Mukhopadhyay B et al.; Starting from D-mannose, D-glucose and L-fucose, the pentasaccharide derivative methyl 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl-(1-->3)-2-O-acetyl-4,6-O-benzylidene-alpha-D-mannopyranosyl-(1-->3)-2-O-acetyl-6-O-benzyl-4-O-(2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl)-alpha-D-mannopyranosyl-(1-->4)-{2-(trimethylsilyl)ethyl 2,3-di-O-benzyl-beta-D-glucopyranosid}uronate was synthesized . This compound with two alpha-mannopyranosyl units was transformed, via Walden inversion and subsequent deprotection, into the alpha-D-glucosamine-type target compound, namely methyl alpha-L-fucopyranosyl-(1-->3)-2-acetamido-2-deoxy-alpha-D-glucopyranosyl-(1-->3)-2-acetamido-2-deoxy-4-O-(alpha-L-fucopyranosyl)-alpha-D-glucopyranosyl-(1-->4)-{2-(trimethylsilyl)ethyl beta-D-glucopyranosid}uronate which is related to the repeating unit of the O-antigen from Shigella dysenteriae type 4.

Int J Antimicrob Agents, 2003 Mar, 21(3), 262 - 6
Comparison of antimicrobial susceptibility between invasive and non-invasive Shigella organisms; Honma Y et al.; Antimicrobial susceptibility was determined for ten strains of Shigella spp . comparing invasive (invasion plasmid containing) and non-invasive members of each strain . The activity of the antimicrobial agents could be classified into three types from the differences between the minimum inhibitory concentration (MIC) for the invasive and non-invasive shigellae . For type 1, there was no difference between the MIC (an MIC gap) for invasive and non-invasive organisms . For type 2, the MIC for the invasive organisms of a strain was higher than that of non-invasive organisms of the strain . In the third type, macrolides taken in by shigellae through the type III secretion apparatus, more effectively inhibited the growth of invasive than non-invasive organisms.

Int J Med Microbiol, 2003 Feb, 292(7-8), 463 - 75
Dissemination of pheU- and pheV-located genomic islands among enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E . coli and their possible role in the horizontal transfer of the locus of enterocyte effacement (LEE); Rumer L et al.; We have recently shown that the locus of enterocyte effacement (LEE) of the bovine enterohemorrhagic E . coli RW1374 (O103:H2) resides within a large pathogenicity island (PAI), integrated in the vicinity of the phenylalanine tRNA gene pheV . Here we describe an additional, but LEE-negative genomic island in RW1374 in the vicinity of another phenylalanine tRNA gene, pheU, the sequence of which is identical to pheV . These two genomic islands revealed identity of the left, but a relative variability of their right end sequences . To investigate the mechanism of LEE-PAI distribution in E . coli, we analysed similar junctions in the pheU/pheV loci of additional EPEC and EHEC strains the LEE location of which had not been determined before . By hybridisation of NotI restriction fragments with probes specific for LEE, pheV locus, and pheU locus, the LEE was found linked to either one of these two loci . The results agreed well with recently published phylogenetic data and indicate that in the clones of diarrheagenic E . coli (Dec) Dec 11 and Dec 12, forming the phylogenetic cluster EPEC 2, and in the strains of the most typical serotypes of the Dec 8, belonging to the phylogenetic cluster EHEC 2, the LEE was linked with pheV and not with the pheU locus as previously assumed . Sequence comparison with other pheU- and pheV-located genomic islands from different E . coli pathotypes (uropathogenic E . coli, septicemic E . coli) as well as from Shigella indicated the same structural features at the junctions . These conserved structures suggested a common DNA cassette, serving as common vehicle for horizontal gene transfer of various PAls . In addition, the elements suggest an origin from a common pheU-located ancestor and integration into the chromosome through site-specific recombination . Our results indicate that pheU/pheV-located genomic islands played an important role in the evolution of several PAls in E . coli and related pathogens.

Euro Surveill . 1996 Jan;1(1):4.
An outbreak of shigellosis in a child care institution in Queluz, Portugal - 1995; Suspiro A et al.; The introduction of shigella into a child care centre carries a high risk of secondary spread from person to person within the centre . We report an outbreak of shigellosis in early 1995 that affected 99 children, 17 of their relations, and seven workers i

J Clin Microbiol, 2003 Mar, 41(3), 926 - 36
stx1c Is the most common Shiga toxin 1 subtype among Shiga toxin-producing Escherichia coli isolates from sheep but not among isolates from cattle; Brett KN et al.; Unlike Shiga toxin 2 (stx(2)) genes, most nucleotide sequences of Shiga toxin 1 (stx(1)) genes from Shiga toxin-producing Escherichia coli (STEC), Shigella dysenteriae, and several bacteriophages (H19B, 933J, and H30) are highly conserved . Consequently, there has been little incentive to investigate variants of stx(1) among STEC isolates derived from human or animal sources . However stx(1OX3), originally identified in an OX3:H8 isolate from a healthy sheep in Germany, differs from other stx(1) subtypes by 43 nucleotides, resulting in changes to 12 amino acid residues, and has been renamed stx(1c) . In this study we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that distinguishes stx(1c) from other stx(1) subtypes . The PCR-RFLP assay was used to study 378 stx(1)-containing STEC isolates . Of these, 207 were isolated from sheep, 104 from cattle, 45 from humans, 11 from meat, 5 from swine, 5 from unknown sources, and 1 from a cattle water trough . Three hundred fifty-five of the 378 isolates (93.9%) also possessed at least one other associated virulence gene (ehxA, eaeA, and/or stx(2)); the combination stx(1), stx(2), and ehxA was the most common (175 of 355 {49.3%}), and 90 of 355 (25.4%) isolates possessed eaeA . One hundred thirty-six of 207 (65.7%) ovine isolates possessed stx(1c) alone and belonged to 41 serotypes . Seventy-one of 136 (52.2%) comprised the common ovine serotypes O5:H(-), O128:H2, and O123:H(-) . Fifty-two of 207 isolates (25.1%) possessed an stx(1) subtype; 27 (51.9%) of these belonged to serotype O91:H(-) . Nineteen of 207 isolates (9.2%) contained both stx(1c) and stx(1) subtypes, and 14 belonged to serotype O75:H8 . In marked contrast, 97 of 104 (93.3%) bovine isolates comprising 44 serotypes possessed an stx(1) subtype, 6 isolates possessed stx(1c), and the remaining isolate possessed both stx(1c) and stx(1) subtypes . Ten of 11 (91%) isolates cultured from meat in New Zealand possessed stx(1c) (serotypes O5:H(-), O75:H8/H40, O81:H26, O88:H25, O104:H(-)/H7, O123:H(-)/H10, and O128:H2); most of these serotypes are commonly recovered from the feces of healthy sheep . Serotypes containing stx(1) recovered from cattle rarely were the same as those isolated from sheep . Although an stx(1c) subtype was never associated with the typical enterohemorrhagic E . coli serogroups O26, O103, O111, O113, and O157, 13 human isolates possessed stx(1c) . Of these, six isolates with serotype O128:H2 (from patients with diarrhea), four O5:H(-) isolates (from patients with hemolytic-uremic syndrome), and three isolates with serotypes O123:H(-) (diarrhea), OX3:H8 (hemolytic-uremic syndrome), and O81:H6 (unknown health status) represent serotypes that are commonly isolated from sheep.

Paediatr Drugs, 2003, 5(3), 151 - 65
Treatment of infectious diarrhea in children; Alam NH et al.; Diarrheal diseases remain an important cause of childhood morbidity and death in developing countries, although diarrheal deaths have significantly declined in recent years, mostly due to successes in the implementation of oral rehydration therapy (ORT), which is the principal treatment modality . Diarrhea may occur for varied reasons; however, most episodes of diarrhea in developing countries are infectious in origin . Three clinical forms of diarrhea (acute watery diarrhea, invasive diarrhea, and persistent diarrhea) have been identified to formulate a management plan . Acute diarrhea may be watery (where features of dehydration are prominent) or dysenteric (where stools contain blood and mucus) . Rehydration therapy is the key to management of acute watery diarrhea, whereas antimicrobial agents play a vital role in the management of acute invasive diarrhea, particularly shigellosis and amebiasis . In persistent diarrhea, nutritional therapy, including dietary manipulations, is a very important aspect in its management, in addition to rehydration therapy . Rehydration may be carried out either by the oral or intravenous route, depending upon the degree of dehydration . Oral rehydration salts (ORS) solution (World Health Organization formula) is recommended for ORT . Intravenous fluid is recommended for initial management of severe dehydration due to diarrhea, followed by ORT with ORS solution for correction of ongoing fluid losses . Antimicrobial therapy is beneficial for cholera and shigellosis . Antiparasitic agents are indicated only if amebiasis and giardiasis are present . Appropriate feeding during diarrhea is recommended for nutritional recovery and to prevent bodyweight loss . Antidiarrheal agents do not provide additional benefit in the management of infectious diarrhea . Although some probiotics have been shown to be beneficial in the treatment of acute diarrhea due to rotavirus, their use in the treatment of diarrhea is yet to be recommended, even in developed countries . The children of developing countries might benefit from zinc supplementation during the diarrheal illness, but its mode of delivery and cost effectiveness are yet to be decided.

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