Shigella Dysenteriae Type Research Articles

The Role of the Complement System in the Pathogenesis of Infectious Forms of Hemolytic Uremic Syndrome.

Hemolytic uremic syndrome (HUS) is an acute disease and the most common cause of childhood acute renal failure. HUS is characterized by a triad of symptoms: microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. In most of the cases, HUS occurs as a result of infection caused by Shiga toxin-producing microbes: hemorrhagic Escherichia coli and Shigella dysenteriae type 1. They account for up to 90% of all cases of HUS. The remaining 10% of cases grouped under the general term atypical HUS represent a heterogeneous group of diseases with similar clinical signs. Emerging evidence suggests that in addition to E. coli and S. dysenteriae type 1, a variety of bacterial and viral infections can cause the development of HUS. In particular, infectious diseases act as the main cause of aHUS recurrence. The pathogenesis of most cases of atypical HUS is based on congenital or acquired defects of complement system. This review presents summarized data from recent studies, suggesting that complement dysregulation is a key pathogenetic factor in various types of infection-induced HUS. Separate links in the complement system are considered, the damage of which during bacterial and viral infections can lead to complement hyperactivation following by microvascular endothelial injury and development of acute renal failure.

A broadly immunogenic polyvalent Shigella multiepitope fusion antigen protein protects against Shigella sonnei and Shigella flexneri lethal pulmonary challenges in mice.

There are no licensed vaccines for Shigella, a leading cause of children's diarrhea and a common etiology of travelers' diarrhea. To develop a cross-protective Shigella vaccine, in this study, we constructed a polyvalent protein immunogen to present conserved immunodominant epitopes of Shigella invasion plasmid antigens B (IpaB) and D (IpaD), VirG, GuaB, and Shiga toxins on backbone protein IpaD, by applying an epitope- and structure-based multiepitope-fusion-antigen (MEFA) vaccinology platform, examined protein (Shigella MEFA) broad immunogenicity, and evaluated antibody function against Shigella invasion and Shiga toxin cytotoxicity but also protection against Shigella lethal challenge. Mice intramuscularly immunized with Shigella MEFA protein developed IgG responses to IpaB, IpaD, VirG, GuaB, and Shiga toxins 1 and 2; mouse sera significantly reduced invasion of Shigella sonnei, Shigella flexneri serotype 2a, 3a, or 6, Shigella boydii, and Shigella dysenteriae type 1 and neutralized cytotoxicity of Shiga toxins of Shigella and Shiga toxin-producing Escherichia coli in vitro. Moreover, mice intranasally immunized with Shigella MEFA protein (adjuvanted with dmLT) developed antigen-specific serum IgG, lung IgG and IgA, and fecal IgA antibodies, and survived from lethal pulmonary challenge with S. sonnei or S. flexneri serotype 2a, 3a, or 6. In contrast, the control mice died, became unresponsive, or lost 20% of body weight in 48 h. These results indicated that this Shigella MEFA protein is broadly immunogenic, induces broadly functional antibodies, and cross-protects against lethal pulmonary challenges with S. sonnei or S. flexneri serotypes, suggesting a potential application of this polyvalent MEFA protein in Shigella vaccine development.

Protective immunization against Enterohemorrhagic Escherichia coli and Shigella dysenteriae Type 1 by chitosan nanoparticle loaded with recombinant chimeric antigens comprising EIT and STX1B-IpaD

Increasing evidence demonstrated that Enterohemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae type 1 (S. dysenteriae1) are considered pathogens, that are connected with diarrhea and are still the greatest cause of death in children under the age of five years, worldwide. EHEC and S. dysenteriae 1 infections can be prevented and managed using a vaccination strategy against pathogen attachment stages. In this study, the chitosan nanostructures were loaded with recombinant EIT and STX1B-IpaD polypeptides. The immunogenic properties of this nano-vaccine candidate were investigated. The EIT and STX1B-IpaD recombinant proteins were heterologous expressed, purified, and confirmed by western blotting. The chitosan nanoparticles, were used to encapsulate the purified proteins. The immunogenicity of recombinant nano vaccine candidate, was examined in three groups of BalB/c mice by injection, oral delivery, and combination of oral-injection. ELISA and antibody titer, evaluated the humoral immune response. Finally, all three mice groups were challenged by two pathogens to test the ability of the nano-vaccine candidate to protect against bacterial infection. The Sereny test in guinea pigs was used to confirm the neutralizing effect of immune sera in controlling S. dysenteriae 1, infections. SDS-PAGE and western blotting, confirmed the presence and specificity of 63 and 27 kDa recombinant EIT and STX1B-IpaD, respectively. The results show that the nanoparticles containing recombinant proteins could stimulate the systemic and mucosal immune systems by producing IgG and IgA, respectively. The challenge test showed that, the candidate nano-vaccine could protect the animal model from bacterial infection. The combination of multiple recombinant proteins, carrying several epitopes and natural nanoparticles could evocate remarkable humoral and mucosal responses and improve the protection properties of synthetic antigens. Furthermore, compared with other available antigen delivery methods, using oral delivery as immune priming and injection as a booster method, could act as combinatorial methods to achieve a higher level of immunity. This approach could present an appropriate vaccine candidate against both EHEC and S. dysenteriae 1.

A story of Shigella vaccine development in ICMR-NICED involving multidimensional approaches

Enteric bacterial infection causes diarrhea throughout the world, especially in developing countries. Shigella is solely answerable toalmost 1.1 million deaths annually in the pediatric population. Vaccine development against diarrheal diseases is always an encouragedconcern. Our laboratory, dedicated to find a possible therapy against shigellosis, is working on a path of various potential methodologiesand immunogens. Over the years, we have concentrated and reported different immunogens with their advantages and drawbacks,ultimately leading us to find the best possible vaccine candidate against bloody diarrhea.The venture started with live attenuated vaccines that protect against multiple serotypes and subtypes of pathogens and found limitedhost-serotype specific immune responses. It was observed that introducing a lipopolysaccharide biosynthesis gene pPR 1347 in Shigelladysenteriae type 1, transformed it into an avirulent organism for candidate vaccine.A mutant strain of Shigella flexneri 2a lacking the RNA-binding protein Hfq was made, leading to increased expression of the type IIIsecretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Suchincreased antigen production and simultaneous attenuation were expected to elicit protective immunity against homologous and alimited number of heterologous serotypes subtypes.Although we formulated the live attenuated vaccine through the introduction of a lipopolysaccharide gene and a mutant lackingRNA binding protein Hfq, but due to lack of heterologous protective efficacy, these were not an ideal vaccine candidate to be madeavailable in the market although they showed a significant amount of immunogenicity. Moreover, live attenuated strains always havea possibility to revert back to its virulent form.Subsequently, monovalent and hexavalent heat-killed immunogens with single and six Shigella serotypes have shown significantprotective efficacy in mice, and rabbit models. Recently we have shown the homologous as well some extent of heterologus protectiveefficacy of heat killed multi-serotype Shigella (HKMS) immunogens in a guinea pig colitis model.A novel formulation for improved immunogen delivery system comprises substantially effective amounts of alginate chitosannanoparticles with OmpA protein of Shigella species. Alginate chitosan nano formulations of OmpA consists essentially of OmpAprotein as conserved active molecule, but efficacy study reveals partial protection efficacy against present circulating Shigella. Furtherimproving the delivery system, we have also formulated a subunit-based vaccine by nanoformulation of ipaC protein of Shigella. Themain drawback of OmpA and ipaC subunit based vaccines are they cannot provide a broad spectrum protection against 50 subtypesand serotypes of Shigella, although they act as a conserved protein in Enterobacteriaceae family, indicating single epitope cannot bethe sole factor associated with the operational protective efficacy.Eventually, our research moved a step ahead and found next-generation outer membrane vesicles (OMVs) based antigens from Shigella.Disruption of tolA, one of the genes of the Tol–Pal system of Shigella membrane, has increased the OMVs release rate by approximately80% higher. Recently we have reported only four serotype-subtype cross-protection among 50 subtypes of circulating Shigella in micemodels. Outer membrane vesicles based immunogen could be a potential cost-effective non-living, next-generation candidate vaccineagainst shigellosis for humans.

Classical- and bioconjugate vaccines: comparison of the structural properties and immunological response.

Glycoconjugate vaccines have been effectively used in humans for about 40 years. The glycoconjugates have substituted plain polysaccharide vaccines thathave many limitations, especially in infants. The covalent linking of protein to carbohydrates has allowed to overcome T-cell-dependent type-2 response of sugars. Glycoconjugates can show improved responses (over plain saccharides) also in elderly and immunocompromised (and depending on the endpoint also in immunocompetent adults), but infants represent the main target of these vaccines because of their unique immune system. Differently from the plain polysaccharide vaccines, the glycoconjugates are also able to induce Immunoglobulin G (IgG) response in infants. Recently, vaccines containing conjugates directly expressed in Escherichia coli (bioconjugates) have been tested in the clinic against Shigella dysenteriae type 1, uropathogenic E. coli, and Streptococcus pneumoniae. Here, we report an overall comparison of classical- and bioconjugate vaccines in terms of the structural properties and the immunological response elicited.

Bacteriostatic and bactericidal activities of Acalypha indica L. plant leave extracts on bacteria shigella disenteriae type 1 in vitro

This study aimed to analyze the bacteriostatic and bactericidal activity of Acalypha indica L. extract against Shigella dysenteriae type 1 bacteria in vitro. This research is a laboratory experimental study consisting of six concentration treatments, namely: (K = 0/ml, P1 = 100/ml, P2 = 200/ml, P3 = 400/ml, P4 = 800/ml, P5 = 1600 mg/ml ), and five replications each. The results showed that the herbal extract of Acalypha indica L. was bacteriostatic (inhibiting) at a concentration of 400 mg/mm, and bactericidal (killing) the bacteria Shigella dysenteriae type 1, which was tested in vitro at a concentration of at least 800 mg/ml. It is necessary to study the effect of Acalypha indica L. herbal extract from Timor in the serum of Rattus norwegicus against bacteria that cause bloody diarrhea (EHEC O157:H7, and Shigella dysenteriae type 1), MRSA bacteria, and other pathogenic bacteria.

Shiga Toxins as Antitumor Tools.

Shiga toxins (Stxs), also known as Shiga-like toxins (SLT) or verotoxins (VT), constitute a family of structurally and functionally related cytotoxic proteins produced by the enteric pathogens Shigella dysenteriae type 1 and Stx-producing Escherichia coli (STEC). Infection with these bacteria causes bloody diarrhea and other pathological manifestations that can lead to HUS (hemolytic and uremic syndrome). At the cellular level, Stxs bind to the cellular receptor Gb3 and inhibit protein synthesis by removing an adenine from the 28S rRNA. This triggers multiple cellular signaling pathways, including the ribotoxic stress response (RSR), unfolded protein response (UPR), autophagy and apoptosis. Stxs cause several pathologies of major public health concern, but their specific targeting of host cells and efficient delivery to the cytosol could potentially be exploited for biomedical purposes. Moreover, high levels of expression have been reported for the Stxs receptor, Gb3/CD77, in Burkitt’s lymphoma (BL) cells and on various types of solid tumors. These properties have led to many attempts to develop Stxs as tools for biomedical applications, such as cancer treatment or imaging, and several engineered Stxs are currently being tested. We provide here an overview of these studies.

Phytochemical constituents of Inula britannica as potential inhibitors of dihydrofolate reductase: A strategic approach against shigellosis

Shigella dysenteriae type 1 is considered as an epidemic in different developing countries, which is responsible for the most severe form of bacterial dysentery. It habitually can develop to the most severe form of dysentery with deadly complications. Development of drugs against this disease is still ongoing. Therefore, we used in silico studies to screen the Inula britannica phytocompounds that are used in traditional Chinese and Kampo Medicines and have activities against different diseases. Spinacetin, eupatin, chrysoeriol and diosmetin were successfully passed through the docking-based screening and absorption, distribution, metabolism, excretion and toxicity (ADMET) filtration. The estimated docking affinities of eupatin, diosmetin, chrysoeriol and spinacetin with Dihydrofolate reductase type 1 (DHFR-1), were −6.5, −6.5, −6.3 and −6.1 kcal/mol, respectively. Which were selected for further investigations based on their favorable ADME/Tox characteristics. Then, the 100 ns molecular dynamics (MD) simulations of apo DHFR, spinacetin-DHFR, eupatin-DHFR, chrysoeriol-DHFR and diosmetin-DHFR complexes were carried out. The RMSD fluctuations of the spinacetin, eupatin, chrysoeriol and diosmetin inside the binding site were explored. Subsequently, the effect of binding Spinacetin, eupatin, chrysoeriol and diosmetin upon the dynamic stability of protein was assessed. Additionally, Principal Component Analysis (PCA) and Hydrogen bond analysis was performed for the apo protein and the protein ligand complexes. The results revealed that chrysoeriol and eupatin has good inhibitory effects against DHFR-1 as treatment for Shigella dysenteriae type when compared to other compounds under study. Hence this study implies that eupatin and chrysoeriol are a significantly potential drug like molecule for the treatment of Shigellosis and must undergo validation through in vivo and in vitro experiments. Communicated by Ramaswamy H. Sarma

Oral immunization of Escherichia albertii strain DM104 induces protective immunity against Shigella dysenteriae type 4 in mouse model.

The recent rise of antibiotic resistance and lack of an effective vaccine make the scenario of shigellosis alarming in developing countries like Bangladesh. In recent years, our group reported the vaccine efficacy of a non-pathogenic Escherichia albertii strain DM104 in different animal models, where an ocularly administered vaccine in the guinea pig eye model against Shigella dysenteriae type 4 challenge showed high protective efficacy and also induced a high titer of serum IgG against S. dysenteriae type 4 whole cell lysate (WCL) and LPS. In this study, we report further evaluation of the non-invasive and non-toxic environmental strain DM104 as a vaccine candidate against S. dysenteriae type 4 in mice model. Oral immunization of live DM104 bacterial strain demonstrated better protective immunity in mice model by showing 90% protection in mice against live S. dysenteriae type 4 lethal dose challenge and by inducing effective humoral and mucosal immune responses.

Isolation and characterization of marine bioluminescent bacteria for toxicity bioassays and biotechnological applications.

Toxic heavy metals pollution posed severe health hazards to the environment and biodiversity. Therefore, the development of rapid and non-invasive bioassays is in the race to monitor toxic chemicals using novel approaches. This study isolated and characterized an intense blue luminescence-producing marine bacteria, Vibrio campbellii STF1, for biosensing applications. Species-level identification of this strain was confirmed based on various phenotypic tests and multilocus sequence approach using 16s rRNA, toxR, and luxA gene sequence analysis. Fatty acid methyl ester analysis revealed the presence of three predominant fatty acids C15:0 anteiso (21.73%), C17:0 anteiso (11.27%), and C19:0 anteiso (9.08%) in STF1. Luciferase enzyme from V. campbellii STF1 was extracted, partially purified, and molecular masses (alpha subunit 40 kDa and beta subunit 37 kDa) were determined by SDS-PAGE gel for in vivo assays. MALDI-TOF-MS analysis of V. campbellii cells' protein extracts showed distinct mass spectral peaks at m/z of 2615, 3948, and 4232 da. V. campbellii STF1 is resistant to heavy metal lead, while other metals such as cadmium, copper, and mercury inhibited its growth and luminescence. Crude ethyl acetate extraction of V. campbellii demonstrated antibacterial activity against Shigella dysenteriae type 5 with a maximum inhibition zone of 27.0±1.0 mm.

Comparison of Immunogenicity and Protective Efficacy of the Intranasal and Intraperitoneal Immunization Routes of <i>Escherichia albertii</i> Strain DM104 in Mouse Model

In recent years, our group isolated the Escherichia albertii strain DM104 and characterized it as a vaccine strain against Shigella dysenteriae type 4 in the guinea pig eye model. Protective efficacy of different routes of immunization such as intranasal, oral, and intrarectal routes were also determined and compared by challenging immunized guinea pigs against live S. dysenteriae. In the current study, we compared the intranasal and intraperitoneal routes of immunizations with the DM104 vaccine strain in mice to understand the better route of administration of the DM104 vaccine and its immunogenicity as well as protective efficacy in mouse model. The results indicate that the immune response elicited by the DM104 strain is strongly dependent on the immunization route, with the intranasal route being more effective than the intraperitoneal route following intraperitoneal live S. dysenteriae challenge. Intranasal immunization yielded 80% protective efficacy in immunized mice whereas, intraperitoneal immunization could not provide any protection. Protection generated by intranasal immunization was accompanied by high titre of anti-whole cell lysate IgG and IgA in DM104 immunized sera compared to sera collected from mice of control group. All these data demonstrate the intranasal route of the vaccine DM104 strain in mouse model to be a better immunization route to protect the animals against live S. dysenteriae challenge.
 Bangladesh J Microbiol, Volume 37 Number 2 December 2020, pp 38-41

Differentiation of stx1A gene for detection of Escherichia coli serotype O157: H7 and Shigella dysenteriae type 1 in food samples using high resolution melting curve analysis.

Escherichia coli serotype O157: H7 and Shigella dysenteriae type 1 as the Shiga toxin‐producing bacteria cause some acute gastrointestinal and extraintestinal diseases such as hemorrhagic uremic syndrome and bloody diarrhea in human. Stx genes are the key virulence factors in these pathogens. The aim of this study was to develop HRMA assay to differentiate stx1A gene for detection of E. coli serotype O157: H7 and Sh. dysenteriae type 1 and determine the prevalence of these pathogens in food samples using this method. PCR‐HRMA assay and gold standard methods have been carried out for identification of pathogens among 135 different food samples. We found HRMA method a sensitive and specific assay (100 and 100%, respectively) for differentiation of stx1A gene, consequently, detection of these pathogens in food samples. Also, the highest prevalence of E. coli serotype O157: H7 and Sh. dysenteriae type 1 harboring stx1A gene was observed in raw milk and vegetable salad samples, respectively. HRMA as a rapid, inexpensive, sensitive and specific method is suggested to be used for differentiation of stx1A gene to detect E. coli serotype O157: H7 and Sh. dysenteriae type 1 as the key pathogens for safety evaluation of food samples.

Characterization and immunogenicity of a Shigella flexneri 2a O-antigen bioconjugate vaccine candidate.

Shigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Vaccination represents a promising preventive measure to fight the burden of the disease, but despite enormous efforts, an efficacious vaccine is not available to date. The use of an innovative biosynthetic Escherichia coli glycosylation system substantially simplifies the production of a multivalent conjugate vaccine to prevent shigellosis. This bioconjugation approach has been used to produce the Shigella dysenteriae type O1 conjugate that has been successfully tested in a phase I clinical study in humans. In this report, we describe a similar approach for the production of an additional serotype required for a broadly protective shigellosis vaccine candidate. The Shigella flexneri 2a O-polysaccharide is conjugated to introduced asparagine residues of the carrier protein exotoxin A (EPA) from Pseudomonas aeruginosa by co-expression with the PglB oligosaccharyltransferase. The bioconjugate was purified, characterized using physicochemical methods and subjected to preclinical evaluation in rats. The bioconjugate elicited functional antibodies as shown by a bactericidal assay for S. flexneri 2a. This study confirms the applicability of bioconjugation for the S. flexneri 2a O-antigen, which provides an intrinsic advantage over chemical conjugates due to the simplicity of a single production step and ease of characterization of the homogenous monomeric conjugate formed. In addition, it shows that bioconjugates are able to raise functional antibodies against the polysaccharide antigen.

Antimicrobial Potential of Epiphytic Bacteria Associated With Seaweeds of Little Andaman, India.

Seaweeds of the intertidal regions are a rich source of surface associated bacteria and are potential source of antimicrobial molecules. In the present study, 77 epiphytic isolates from eight different algae collected from Little Andaman were enumerated. On testing for their antimicrobial activities against certain pathogens twelve isolates showed positive and six of them showed significant antimicrobial inhibition zone against Shigella boydii type 1, Shigella flexneri type 2a, Shigella dysenteriae type 5, Enterotoxigenic Escherichia coli O115, Enteropathogenic E. coli serotype O114, Vibrio cholera; O1 Ogawa, Aeromonas hydrophila, Klebsiella pneumoniae, Staphylococcus aureus. Based on the activity these six isolates (G1C, G2C, G3C, UK, UVAD, and Tor1) were identified by 16S rRNA gene sequence and were found to belong to the phyla Firmicutes and Proteobacteria. Purified antimicrobial compounds obtained from these isolates were identified by GC-MS. Furan derivatives were identified from G2C Pseudomonas stutzeri KJ849834, UVAD Alcanivorax dieselolei KJ849833, UK Vibrio sp. KJ849837, Tor1 Exiguobacterium profundum KJ849838. While 2-Pyrrolidinone, Phenol, 2, 4-bis (1, 1-dimethylethyl) were from G3C Vibrio owensii KJ849836 and (1-Allylcyclopropyl) methanol from the extracts of G1C Bacillus sp. KJ849835. The results of the present study shows that these six potent isolates isolated from the seaweeds are found to be a source of antimicrobial compounds.

Structure elucidation of the O-specific polysaccharide by NMR spectroscopy and selective cleavage and genetic characterization of the O-antigen of Escherichia albertii O5

The O-specific polysaccharide (O-antigen) was obtained by mild acid degradation of the lipopolysaccharide of Escherichia albertii O5 (strain T150248) and studied by sugar analysis, selective cleavages of glycosidic linkages, and 1D and 2D 1H and 13C NMR spectroscopy. Partial solvolysis with anh (anhydrous) CF3CO2H and hydrolysis with 0.05 M CF3CO2H cleaved predominantly the glycosidic linkage of β-GalpNAc or β-Galf, respectively, whereas the linkages of α-GlcpNAc and β-Galp were stable. Mixtures of the corresponding tri- and tetra-saccharides thus obtained were studied by NMR spectroscopy and high-resolution ESI MS. The following new structure was established for the tetrasaccharide repeat (O-unit) of the O-polysaccharide: →4)-α-d-GlcpNAc-(1 → 4)-β-d-Galp6Ac-(1 → 6)-β-d-Galf-(1 → 3)-β-d-GalpNAc-(1→where the degree of O-acetylation of d-Galp is ∼70%. The O-polysaccharide studied has a β-d-Galp-(1 → 6)-β-d-Galf-(1 → 3)-β-d-GalpNAc trisaccharide fragment in common with the O-polysaccharides of E. albertii O7, Escherichia coli O124 and O164, and Shigella dysenteriae type 3 studied earlier. The orf5-7 in the O-antigen gene cluster of E. albertii O5 are 47%, 78%, and 75% identical on the amino acid level to genes for predicted enzymes of E. albertii O7, including Galp-transferase wfeS, UDP-d-Galp mutase glf, and Galf-transferase wfeT, respectively, which are putatively involved with the synthesis of the shared trisaccharide fragment of the O-polysaccharides. The occurrence upstream of the O-antigen gene cluster of a 4-epimerase gene gnu for conversion of undecaprenyl diphosphate-linked d-GlcNAc (UndPP-d-GlcNAc) into UndPP-d-GalNAc indicates that d-GalNAc is the first monosaccharide of the O-unit, and hence the O-units are interlinked in the O-polysaccharide of E. albertii O5 by the β-d-GalpNAc-(1 → 4)-α-d-GlcpNAc linkage.

Effects of Oxalis barrelieri L. (Oxalidaceae) aqueous extract on diarrhea induced by Shigella dysenteriae type 1 in rats.

Aim Oxalis barrelieri is a medicinal plant commonly used in Cameroon, for the treatment of many diarrheal diseases. The antibacterial properties of O barrelieri aqueous extract (WOb) against Shigella dysenteriae type 1 were investigated in vitro and in vivo.Methods Antibacterial activity was evaluated in vitro by disc diffusion method and by macrodilution method. S dysenteriae type 1 at a dose of 1.2 × 109 CFU was administrated orally to rats to induce shigellosis. For 6 consecutive days, diarrheic rats were treated with O barrelieri aqueous extract (50 and 100 mg/kg BW) or norfloxacin (20 mg/kg BW). The diarrheal stool weight and S dysenteriae type 1 density were assessed during the treatment period, and death rate recorded. Nitric oxide production in blood and in colonic homogenate and blood parameters were assessed, and the histological section of the colon was performed in the survivors.ResultsThe minimal inhibitory concentration and minimal bactericidal concentration of WOb were, respectively, 6 mg/mL and 25 mg/mL. The mean minimal bactericidal concentration/minimal inhibitory concentration ratio for WOb against S dysenteriae type 1 was high (˃4); WOb could be classified as a bacteriostatic drug. WOb significantly (P < .01) reduced bacterial density and diarrheal stool weight. WOb decreased nitric oxide production (P < .01) in the large intestine and protected the mucosa of the colon from bacterial destruction.ConclusionThe results suggest that O barrelieri aqueous extract possesses bacteriostatic and antidiarrheal activities and reduces damages caused to intestinal mucosa barrier by pathogenic mechanisms of Shigella. This extract could be used as an alternative therapeutic for infectious diarrhea.

Detection of Shigella dysenteriae Type 1 in Milk by PCR

&lt;p&gt;Shigellosis is severest form of bacillary dysentery, a disease limited to humans and certain other primates. Conventional microbiological techniques used for the isolation and biochemical identification of microbes are time-consuming as well as labour intensive. Due to the fastidious nature of &lt;em&gt;Shigella&lt;/em&gt; and the lengthy culture time, a rapid and sensitive system for the detection of bacteria is highly desirable. Nucleic acid-based techniques have enormous potential in the detection of biological weapons because of their specificity, sensitivity, and the speed with which results can be obtained. The application of polymerase chain reaction in sensitive detection of bacterial pathogens in direct food samples is largely affected by the quality of the template DNA. To overcome the shortcomings of conventional methods, we have defined simple method of sample preparation that facilitates PCR based detection of &lt;em&gt;Shigella dysenteriae&lt;/em&gt; in milk. In present study, gene specific primers were designed to detect &lt;em&gt;stxA&lt;/em&gt; gene.&lt;em&gt; &lt;/em&gt;The genomic DNA extraction from the spiked milk was carried out by proteinase K, lysozyme, chloroform-isoamyl alcohol and heat treatments. The optimized extraction of DNA by addition of chloroform-isoamyl alcohol proved to be the most satisfactory amongst all, with limit of detection of 2.0 x 10&lt;sup&gt;3 &lt;/sup&gt;cfu/ reaction and was completed in 4h.&lt;/p&gt;&lt;p&gt; &lt;/p&gt;

Remediation of intramacrophageal Shigella dysenteriae type 1 by probiotic lactobacilli isolated from human infants' stool samples.

Background & objectives:Shigella dysenteriae is one of the most virulent pathogens causing bacillary dysentery and is responsible for high mortality in infants. To reduce the load of antibiotic therapy for treating shigellosis, this study was carried out to assess the ex vivo effect of novel probiotic lactobacilli, isolated from infant's stool samples, on killing S. dysenteriae type 1 residing in the rat macrophages.Methods:Stool samples from infants were collected, processed for the isolation of lactobacilli and screened for the probiotic attributes (acid tolerance, bile tolerance, ability to adhere intestinal cells and anti-S. dysenteriae activity). The effect of cell-free supernatant of lactobacilli on Shigella- infected macrophages in terms of phagocytic ability, extent of lipid peroxidation, nitrite, superoxide dismutase and glutathione levels was evaluated.Results:Based on the probiotic attributes, three lactobacilli were isolated from the stool samples of infants. Using classical and molecular tools, these isolates were identified as Lactobacillus pentosus, L. Paraplantarum and L. rhamnosus. All the three lactobacilli had the ability to kill intramacrophage S. dysentriae type 1. The anti-Shigella activity of the probiotic lactobacilli was attributed to increased antioxidative ability and decreased free radical production by the infected macrophages.Interpretation & conclusions:Probiotic cocktail of L. pentosus, L. paraplantarum and L. rhamnosus showed ex vivo killing of S. dysenteriae residing inside the rat macrophages significantly. This cocktail has the potential to be used as a natural alternative for treating S. dysenteriae infection, especially in infants, however, further studies need to be done to confirm these finding in vivo.

Immunization with r-Lactococcus lactis expressing outer membrane protein A of Shigella dysenteriae type-1: evaluation of oral and intranasal route of administration.

To evaluate the comparative immunogenic potential of food grade Lactococcus lactis expressing outer membrane protein A (OmpA) of Shigella dysenteriae type-1 (SD-1) when administered either orally or intranasally. OmpA of SD-1 was cloned and expressed first in Escherichia coli and then in L. lactis. Presence of recombinant gene was confirmed by restriction enzyme digestion and immunoblot analysis. Using immobilized metal affinity chromatography, OmpA was purified from recombinant E.coliBL21 (DE3) and subcutaneously administered in BALB/c mice. Detection of OmpA-specific IgG antibodies by enzyme-linked immunosorbent assay (ELISA) confirmed the immunogenicity of OmpA. In order to establish r-L. lactis as a mucosal delivery vehicle, it was administered orally and nasally in BALB/c mice. Serum IgG and faecal IgA were assessed through ELISA to compare the relative potential of immunization routes and immunogenic potential of r-L. lactis. Immunization via the oral route proved superior to intranasal exposure. Recombinant L. lactis expressing OmpA of SD-1 was found to be immunogenic. Oral administration of r-L. lactis elicited higher systemic and mucosal immune response when compared with the nasal route. Using food grade recombinant L. lactis has implications in the development of a prophylactic against multidrug-resistant Shigella, which can be used as a prospective vaccine candidate. Evaluating mucosal routes of immunization demonstrated that the oral route of administration elicited better immune response against OmpA of Shigella.

Identification of serotypes and virulence markers (stx) of Escherichia coli isolated from patients with diarrhea in Shiraz, Iran

BackgroundIn developing countries diarrheal infections with bacterial species especially enterohemorrhagic Escherichia coli (EHEC) cause severe diseases including hemorrhagic colitis, hemolytic uremic syndrome and mortality in children less than five years of age. Such life-threatening infections are due to Shiga toxin (Stx) produced by EHEC including stx1 and stx2. stx1 is identical to shiga toxin produced by Shigella dysenteriae type1. However, stx2 is different from Stx1 in immunological properties, but similar to Stx1 in biological characteristics. Both of these toxins are encoded by stx genes of lysogenic bacteriophage (Stx phage) integrated into the genome of EHEC. AimsThe aim of this study was to determine the prevalence and distribution of Stx toxins in O157:H7E.coli and non-O157:H7E. coli isolated from patients with diarrhea from 2013 to 2014 in Shiraz, Iran. Methods and materialsStool samples from 1050 children and adults patients collected from June 2014 to June 2015 were investigated for Shiga toxigenic E. coli by conventional and molecular methods. ResultsOf 1050 diarrheal specimens, 306 isolates (29.1%) was diagnosed as E. coli. Gene analysis for virulence factors showed that 38(12.4%) isolates carried Stx1, 57(18.6%) Stx2 and 13 (4.2%) harbored both Stx1 and Stx2. ConclusionsThis study showed that ETEC was potential pathogen in children aged less than five years and the relatively high prevalence of Stx1 and Stx2 producing ETEC in our region. Moreover, the higher detection rate of Stx2 indicated the important role of this gene in diarrheal diseases