Unlocking the potential of Extensin Signal peptide and Elastin-like polypeptide tag fused to Shigella dysenteriae's IpaDSTxB to improve protein expression and purification in Nicotiana tabacum and Medicagosativa.

Plants are often seen as a potent tool in the recombinant protein production industry. However, unlike bacterial expression, it is not a popular method due to the low yield and difficulty of protein extraction and purification. Therefore, developing a new high efficient and easy to purify platform is crucial. One of the best approaches to make extraction easier is to utilize the Extensin Signal peptide (EXT) to translocate the recombinant protein to the outside of the cell, along with incorporating an Elastin-like polypeptide tag (ELP) to enhance purification and accumulation rates. In this research, we transiently expressed Shigella dysenteriae's IpaDSTxB fused to both NtEXT and ELP in both Nicotiana tabacum and Medicago sativa. Our results demonstrated that N. tabacum, with an average yield of 6.39 ng/µg TSP, outperforms M. sativa, which had an average yield of 3.58 ng/µg TSP. On the other hand, analyzing NtEXT signal peptide indicated that merging EXT to the constructs facilitates translocation of IpaDSTxB to the apoplast by 78.4% and 65.9% in N. tabacum and M. sativa, respectively. Conversely, the mean level for constructs without EXT was below 25% for both plants. Furthermore, investigation into the orientation of ELP showed that merging it to the C-terminal of IpaDSTxB leads to a higher accumulation rate in both N. tabacum and M. sativa by 1.39 and 1.28 times, respectively. It also facilitates purification rate by over 70% in comparison to 20% of the 6His tag. The results show a highly efficient and easy to purify platform for the expression of heterologous proteins in plant.

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.

Impact of insertion sequences on convergent evolution of Shigella species.

Shigella species are specialised lineages of Escherichia coli that have converged to become human-adapted and cause dysentery by invading human gut epithelial cells. Most studies of Shigella evolution have been restricted to comparisons of single representatives of each species; and population genomic studies of individual Shigella species have focused on genomic variation caused by single nucleotide variants and ignored the contribution of insertion sequences (IS) which are highly prevalent in Shigella genomes. Here, we investigate the distribution and evolutionary dynamics of IS within populations of Shigella dysenteriae Sd1, Shigella sonnei and Shigella flexneri. We find that five IS (IS1, IS2, IS4, IS600 and IS911) have undergone expansion in all Shigella species, creating substantial strain-to-strain variation within each population and contributing to convergent patterns of functional gene loss within and between species. We find that IS expansion and genome degradation are most advanced in S. dysenteriae and least advanced in S. sonnei; and using genome-scale models of metabolism we show that Shigella species display convergent loss of core E. coli metabolic capabilities, with S. sonnei and S. flexneri following a similar trajectory of metabolic streamlining to that of S. dysenteriae. This study highlights the importance of IS to the evolution of Shigella and provides a framework for the investigation of IS dynamics and metabolic reduction in other bacterial species.

Molecular Mechanisms of Shigella Pathogenesis; Recent Advances.

Shigella species are the main cause of bacillary diarrhoea or shigellosis in humans. These organisms are the inhabitants of the human intestinal tract; however, they are one of the main concerns in public health in both developed and developing countries. In this study, we reviewed and summarised the previous studies and recent advances in molecular mechanisms of pathogenesis of Shigella Dysenteriae and non-Dysenteriae species. Regarding the molecular mechanisms of pathogenesis and the presence of virulence factor encoding genes in Shigella strains, species of this bacteria are categorised into Dysenteriae and non-Dysenteriae clinical groups. Shigella species uses attachment, invasion, intracellular motility, toxin secretion and host cell interruption mechanisms, causing mild diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome diseases in humans through the expression of effector delivery systems, protein effectors, toxins, host cell immune system evasion and iron uptake genes. The investigation of these genes and molecular mechanisms can help us to develop and design new methods to detect and differentiate these organisms in food and clinical samples and determine appropriate strategies to prevent and treat the intestinal and extraintestinal infections caused by these enteric pathogens.

Effect of Chestnut (Castanea Mollissima Blume) Bur Polyphenol Extract on Shigella dysenteriae: Antibacterial Activity and the Mechanism.

Shigella dysenteriae is a highly pathogenic microorganism that can cause human bacillary dysentery by contaminating food and drinking water. This study investigated the antibacterial activity of chestnut bur polyphenol extract (CBPE) on S. dysenteriae and the underlying mechanism. The results showed that the minimum inhibitory concentration (MIC) of CBPE for S. dysenteriae was 0.4 mg/mL, and the minimum bactericidal concentration (MBC) was 1.6 mg/mL. CBPE treatment irreversibly disrupted cell morphology, decreased cell activity, and increased cell membrane permeability, cell membrane depolarization, and cell content leakage of S. dysenteriae, indicating that CBPE has obvious destructive effects on the cell membrane and cell wall of S. dysenteriae. Combined transcriptomic and metabolomics analysis revealed that CBPE inhibits S. dysenteriae by interfering with ABC protein transport, sulfur metabolism, purine metabolism, amino acid metabolism, glycerophospholipid metabolism, and some other pathways. These findings provide a theoretical basis for the prevention and treatment of S. dysenteriae infection with extract from chestnut burs.

The heme-binding protein PhuS transcriptionally regulates the Pseudomonas aeruginosa tandem sRNA prrF1,F2 locus.

Pseudomonas aeruginosa is an opportunistic pathogen requiring iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the nonredundant heme assimilation system and Pseudomonas heme uptake (Phu) systems. Heme transported by either the heme assimilation system or Phu system is sequestered by the cytoplasmic protein PhuS. Furthermore, PhuS has been shown to specifically transfer heme to the iron-regulated heme oxygenase HemO. As the PhuS homolog ShuS from Shigella dysenteriae was observed to bind DNA as a function of its heme status, we sought to further determine if PhuS, in addition to its role in regulating heme flux through HemO, functions as a DNA-binding protein. Herein, through a combination of chromatin immunoprecipitation-PCR, EMSA, and fluorescence anisotropy, we show that apo-PhuS but not holo-PhuS binds upstream of the tandem iron-responsive sRNAs prrF1,F2. Previous studies have shown the PrrF sRNAs are required for sparing iron for essential proteins during iron starvation. Furthermore, under certain conditions, a heme-dependent read through of the prrF1 terminator yields the longer PrrH transcript. Quantitative PCR analysis of P. aeruginosa WT and ΔphuS strains shows that loss of PhuS abrogates the heme-dependent regulation of PrrF and PrrH levels. Taken together, our data show that PhuS, in addition to its role in extracellular heme metabolism, also functions as a transcriptional regulator by modulating PrrF and PrrH levels in response to heme. This dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network that is critical for P. aeruginosa adaptation and virulence within the host.

Chemical Synthesis and Antigenicity Evaluation of Shigella dysenteriae Serotype 10 O-Antigen Tetrasaccharide Containing a (S)-4,6-O-Pyruvyl Ketal.

Shigella is the second most common etiologic pathogen responsible for childhood acute diarrhea. An anti-Shigella vaccine is still eagerly awaited due to the increasing drug resistance of this pathogen. The Shigella O-antigen is a promising vaccine target. To identify the immune epitopes of the glycan, the first total synthesis of Shigella dysenteriae serotype 10 O-antigen tetrasaccharide containing a (S)-4,6-O-pyruvyl ketal was completed. The 1,2-trans-ß-glycosylation & C2 epimerization and conformational locking strategies facilitated the construction of two 1,2-cis-ß-glycosidic linkages. The reactivities of both the glycosyl donor and acceptor were improved by adding electron-donating benzyl groups, enabling an efficient assembly of the tetrasaccharide. The (S)-4,6-O-pyruvyl ketal was introduced at the final stage due to its influence on the glycosylation stereospecificity and efficiency. In addition, (R)-4,6-O-pyruvylated and nonpyruvylated tetrasaccharides and three further fragments were synthesized. Glycan microarray screening revealed that the tetrasaccharide repeating unit is the key antigenic epitope of the O-antigen. Moreover, the (S)-4,6-O-pyruvyl ketal is an essential structural feature of this antigen for designing carbohydrate-based vaccines against S. dysenteriae serotype 10. The comparison of the (S)-4,6-O-pyruvylated glycan and its (R)-epimer will set an example for biological evaluation of other bacterial glycans containing pyruvyl ketals.

Epidemic and molecular characterization of fluoroquinolone-resistant Shigella dysenteriae 1 isolates from calves with diarrhea.

BACKGROUND: The widespread distribution of antimicrobial-resistant Shigella has become a recurrent challenge in many parts of the developing world. Previous studies indicate that the host of Shigella has expanded from humans to animals. This study aimed to investigate the prevalence of fluoroquinolone resistance and associated molecular characterization of S. dysenteriae 1 isolated from calves. RESULTS: All 38 unduplicated S. dysenteriae 1 isolates were collected from calves in Gansu Province from October 2014 to December 2016. According to MLST and PFGE analysis, these isolates were separated into 4 and 28 genotypes, respectively. The most common STs identified were ST228 (34.21%, 13/38) and ST229 (39.47%, 15/38), which were first found in the present study. All isolates harbored virulence genes, and the incidence of the seven virulence genes were ipaH (100%), ipaBCD (92.11%), stx (73.68%), ial (57.89%), sen (28.95%), set1A and set1B (0%). According to the results of antimicrobial susceptibilities, 76.32% (29/38) were resistant to fluoroquinolone and showed multidrug resistance. In a study on the polymorphism of quinolone resistance-determining region (QRDR) of gyrA/B and parC/E genes, we identified two mutations in gyrA (Ser83 → Leu and Asp87 → Asn) and parC (Ser80 → Ile and Ser83 → Leu), respectively. Among them, 55.17% (16/29) of resistant strains had the gyrA point mutations (Ser83 → Leu) and parC point mutation (Ser83 → Leu). Moreover, 41.38% (12/29) of isolates had all five point mutations of gyrA and parC. In addition, the prevalence of the plasmid-mediated quinolone resistance (PMQR) determinant genes was also investigated. All 29 fluoroquinolone-resistant isolates were positive for the aac (6')-Ib-cr gene but negative for qepA, except for SD001. In addition, only 6 (20.69%, 6/29) isolates harbored the qnr gene, including two with qnrB (6.90%, 2/29) and four with qnrS (13.79%, 4/29). CONCLUSION: Given the increased common emergence of multidrug resistant isolates, uninterrupted surveillance will be necessary to understand the actual epidemic burden and control this infection.

Complete genome analysis of the newly isolated Shigella sonnei phage vB_SsoM_Z31.

This work describes the characterization and genome annotation of the newly isolated lytic phage vB_SsoM_Z31 (referred to as Z31), isolated from wastewater samples collected in Dalian, China. Transmission electron microscopy revealed that phage Z31 belongs to the family Myoviridae, order Caudovirales. This phage specifically infects Shigella sonnei, Shigella dysenteriae, and Escherichia coli. The genome of the phage Z31 is an 89,355-bp-long dsDNA molecule with a G+C content of 38.87%. It was predicted to contain 133 ORFs and encode 24 tRNAs. No homologs of virulence factor genes or antimicrobial resistance genes were found in this phage. Based on the results of nucleotide sequence alignment and phylogenetic analysis, phage Z31 was assigned to the genus Felixounavirus, subfamily Ounavirinae.

Activity of the lyases LysSSE1 and HolSSE1 against common pathogenic bacteria and their antimicrobial efficacy in biofilms.

Bacillary dysentery is a common foodborne disease with an exaggerated mortality rate because of Shigella infection. With the increasing severity of Shigella infection, lyase has been considered as the most promising alternative to antimicrobial agents, owing to the emergence of resistant bacteria and the difficulty in disrupting and eliminating bacterial biofilms. In this study, we cloned and characterised HolSSE1 and LysSSE1, holin, and lysozyme from the S. dysenteriae phage SSE1 with extended bacterial host range against common gram-negative and gram-positive bacteria. In addition, the efficacy of HolSSE1 and LysSSE1 in removing bacterial biofilms was observed on polystyrene surfaces. Moreover, synergistic bacteriostasis was observed when they were used together. Alignment and structural model analysis showed that both HolSSE1 and LysSSE1 are T4 phage proteins that have not yet been identified. Therefore, HolSSE1 and LysSSE1 can be promising biocontrol agents for the prevention and treatment of various pathogenic infections.

An in-vitro study on a novel six-phage cocktail against multi-drug resistant-ESBL Shigella in aquatic environment.

Shigella spp. are water-borne pathogens responsible for mild to severe cases bacilli dysentery all around the world known as Shigellosis. The progressively increasing of antibiotic resistance among Shigella calls for developing and establishing novel alternative therapeutic methods. The present study aimed to evaluate a novel phage cocktail of lytic phages against extended spectrum beta lactamase isolates of Shigella species in an aquatic environment. The phage cocktail containing six novel Shigella specific phages showed a broad host spectrum. The cocktail was very stable in aquatic environment. The cocktail resulted in about 99% decrease in the bacterial counts in the contaminated water by several species and strains of Shigella such as Shigella sonnei, Shigella flexneri and Shigella dysenteriae. Achieving such a high efficiency in this in-vitro study demonstrates a high potential for in-vivo and in-situ application of this phage cocktail as a bio-controlling agent against Shigella spp. contamination and infections.

Antimicrobial and antioxidant activity of catechin-3-o-rhamnoside isolated from the stem bark of Lannea kerstingii Engl. and K. Krause (Anacardiaceae).

Various epidemiological researches have shown that consumption of vegetables and fruits are essential to maintain health and prevent diseases but the emergence of more and more drug resistance bacteria has led to high mortality. Thus the study of the antimicrobial and antioxidant activities of a flavonoid (Catechin-3-o-rhamnoside) isolated for the first time from Lannea kerstingii. Catechin-3-o-rhamnoside was isolated using dry vacuum liquid chromatography. It was characterized using 1H-NMR, 13C-NMR and 2D NMR spectra. The antimicrobial activity was determined using agar diffusion and broth dilution method. Antioxidant activity was determined through reaction of the compound with DPPH radical. The compound was active against, Methicillin Resistant Staphylococcus aureus, S. aureus, B. subtilis, E. coli, K. pneumoniae, S. typhi, S. dysentariae, C. albicans and C. tropicalis with zone of inhibition ranging from 22.0±0.1 to 35.0±0.2mm and inactive against vancomycin resistant enterococci, Proteus mirabilis and C. ulcerans. The MIC ranged from 6.25 to 12.5µg/ml while the MBC/MFC ranged from 12.5 to 50.0µg/ml. The compound showed a high radical scavenging activity with EC50 of 46.87µg/ml. These results show a potential lead drug for resistant bacteria and natural antioxidants.

Regulation of OmpA Translation and Shigella dysenteriae Virulence by an RNA Thermometer.

RNA thermometers are cis-acting riboregulators that mediate the posttranscriptional regulation of gene expression in response to environmental temperature. Such regulation is conferred by temperature-responsive structural changes within the RNA thermometer that directly result in differential ribosomal binding to the regulated transcript. The significance of RNA thermometers in controlling bacterial physiology and pathogenesis is becoming increasingly clear. This study combines in silico, molecular genetics, and biochemical analyses to characterize both the structure and function of a newly identified RNA thermometer within the ompA transcript of Shigella dysenteriae First identified by in silico structural predictions, genetic analyses have demonstrated that the ompA RNA thermometer is a functional riboregulator sufficient to confer posttranscriptional temperature-dependent regulation, with optimal expression observed at the host-associated temperature of 37°C. Structural studies and ribosomal binding analyses have revealed both increased exposure of the ribosomal binding site and increased ribosomal binding to the ompA transcript at permissive temperatures. The introduction of site-specific mutations predicted to alter the temperature responsiveness of the ompA RNA thermometer has predictable consequences for both the structure and function of the regulatory element. Finally, in vitro tissue culture-based analyses implicate the ompA RNA thermometer as a bona fide S. dysenteriae virulence factor in this bacterial pathogen. Given that ompA is highly conserved among Gram-negative pathogens, these studies not only provide insight into the significance of riboregulation in controlling Shigella virulence, but they also have the potential to facilitate further understanding of the physiology and/or pathogenesis of a wide range of bacterial species.

Lactobacillus acidophilus attenuates toxin production by Vibrio cholerae and shigella dysenteriae following intestinal epithelial cells infection.

AIMS: The main objective of the present study was to assess and compare the safety and inhibitory efficacy of Lactobacillus acidophilus against cholera toxin and shigatoxin production by measuring CTX-B and Stx1 expression level in Caco-2 cells exposed to Vibrio cholerae (as a non-invasive small intestine pathogens and Shigella dysenteriae (as an invasive colon pathogen). METHODS: Caco-2 cells were incubated with L. acidophilus 2 h before infection by V. cholerae and S. dysenteriae. Following RNA extraction and cDNA synthesis, relative toxins mRNA levels were determined according to a comparative critical threshold (Ct) real-time PCR. L. acidophilus didn't show any cytotoxic effect on Caco-2 cells. RESULTS: L. acidophilus revealed a protective effect for Caco-2 cells against S. dysenteriae and V. cholera by 51% and 57%, respectively, which was determined by MTT assay and further confirmed by morphological examination. Pretreatment of Caco-2 cells with L. acidophilus prior to exposure to V. cholerae, attenuated the CTX-B expression in V. cholerae to about 1.76 folds. Expression of Stx1 by S. dysenteriae was also down-regulated to 1.6 folds following pretreatment of Caco-2 cells by L. acidophilus. No significant difference was observed in the attenuator role of L. acidophilus in toxin production by S. dysenteriae as a colon-invasive bacterium, compared with V. cholerae, the non-invasive pathogen of small intestine. CONCLUSIONS: The results of the present study suggest that L. acidophilus is safe with protective effect for human epithelial colorectal cells, and is effective enough to be applied as a supplementary treatment for attenuation of toxin production in acute infectious diarrhea caused by V. cholerae and S. dysenteriae.

Development of quinolone resistance and prevalence of different virulence genes among Shigella flexneri and Shigella dysenteriae in environmental water samples.

The purpose of this study was to find out the mechanism of quinolone resistance in Shigella sp. isolated from environmental water samples from various parts of Kolkata, India. Out of 196 Shigella sp. isolated from 2014 to 2017, we selected 32 Shigella isolates for antimicrobial susceptibility tests. The minimum inhibitory concentrations (MIC) for quinolones ranged from 30 to 50 µg ml-1 for ofloxacin, 5-20 µg ml-1 for ciprofloxacin and 20-30 µg ml-1 for norfloxacin. A few amino acid changes were found in quinolone resistance determining region (QRDR) of gyrA. Mutations in gyrA lead to a higher increment of MIC of quinolones. Among the plasmid-mediated (PMQR) quinolone resistance genes investigated, qnrB and aac(6')-lb-cr genes were found in all isolates. qnrA and qnrS were found in 25% and 62% of the isolates, respectively. ipaH gene was found in all of the isolates followed by the presence of other virulence genes ial, sen and stx1. Almost all the isolates having high MICs showed efflux pump activity in drug accumulation assay. All the mechanisms may or may not be present in a single strain. Several types of efflux pumps, presence of PMQR genes and mutations in drug target site of QRDR region may play the crucial role for resistance in our isolates.

SERS-based immunocapture and detection of pathogenic bacteria using a boronic acid-functionalized polydopamine-coated Au@Ag nanoprobe.

A surface-enhanced Raman scattering (SERS)-based immunocapture nanoprobe is described for the detection of pathogenic bacteria. The probe uses boronic acid-functionalized polydopamine-coated Au@Ag nanoparticles as an advanced SERS nanotag. Modified magnetic IgG@Fe3O4 nanoparticles are used for magnetic separation. Au@Ag@PDA nanoparticles, where PDA stands for polydopamine, were functionalized with boronic acid to bind to pathogenic bacteria and induce signal amplification. The Raman signal is amplified 108 times when the SERS tag binds the surface of bacteria. The SERS spectra exhibit fingerprint-like patterns that enable bacterial classification. The results of principal component analysis (PCA) and hierarchical cluster analysis (HCA) of the spectral regions were compared. The bacterial surface protein and glycan signals (1300-1450 cm-1) were the best regions for bacterial classification. Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Pseudomonas aeruginosa, and Klebsiella pneumonia were successfully classified by this method. The lowest detection limit was 10 colonies/mL (CFU·mL). The assay can be completed within 30 min. Conceivably, this method may be extended to the quantitative detection or classification of bacteria under various other conditions. Graphical abstract Schematic representation of immunocapture and detection of pathogenic bacteria using boronic acid-functionalized polydopamine-coated Au@Ag nanoprobe through the bacterial surface protein and glycan signals. Green arrow: laser; black arrow: SERS; red ball: bacteria; grey ball: IgG@Fe3O4; golden ball: boronic acid-functionalized Au@Ag@PDA.

Shiga Toxin Induces Lipid Compression: A Mechanism for Generating Membrane Curvature.

Biomembranes are hard to compress laterally, and membrane area compressibility has not been associated with biological processes. Using X-ray surface scattering, we observed that bacterial Shiga toxin compresses lipid packing in a gel phase monolayer upon binding to its cellular receptor, the glycolipid Gb3. This toxin-induced reorganization of lipid packing reached beyond the immediate membrane patch that the protein was bound to, and linkers separating the Gb3 carbohydrate and ceramide moieties modulated the toxin's capacity to compress the membrane. Within a natural membrane, asymmetric compression of the toxin-bound leaflet could provide a mechanism to initiate narrow membrane bending, as observed upon toxin entry into cells. Such lipid compression and long-range membrane reorganization by glycolipid-binding proteins represent novel concepts in membrane biology that have direct implications for the construction of endocytic pits in clathrin-independent endocytosis.

Isolation, characterization, and PCR-based molecular identification of a siphoviridae phage infecting Shigella dysenteriae.

BACKGROUND: Shigella dysenteriae is one of the members of Shigella genus which was the main responsible of different Shigellosis outbreaks worldwide. The increasing consumption of antibiotics has led to the emergence and spreading of antibiotic-resistant strains. Therefore, finding new alternatives for infection control is essential, one of which is using bacteriophages. MATERIALS AND METHODS: Lytic bacteriophage against Shigella dysenteriae was isolated from petroleum refinery wastewater. Phage morphological and genetic characteristics were studied using TEM, and sequencing, respectively. In addition, the genome size was estimated, and phage resistance to different temperatures and pH, host range, adsorption rate, and one-step growth were investigated. RESULTS: According to the morphology and genetic results, this phage was named vB-SdyS-ISF003. Sequencing of the PCR products revealed that the vB-SdyS-ISF003 phage belongs to the species T1virus, subfamily Tunavirinae of family Siphoviridae. This was the first detected bacteriophage against S. dysenteriae, which belongs to the family Siphoviridae. In addition, its host range was limited to S. dysenteriae. The genome size was about 62 kb. vB-SdyS-ISF003 phage has a number of desirable characteristics including the limited host range to S. dysenteriae, very short connection time, a relatively wide range of temperature tolerance -20 to 50 °C, pH tolerance of 7-9 without significant reduction in the phage titer. CONCLUSION: vB-SdyS-ISF003 is a novel virulent T1virus phage and has the appropriate potential for being used in bio controlling of S. dysenteriae in different condition.

Immunological detection assays for recombinant Shiga toxin & Shigella dysenteriae.

Background & objectives: : Shiga toxin (Stx) is produced by Shigella dysenteriae, a Gram-negative, facultative anaerobic bacillus that causes shigellosis, haemolytic uraemic syndrome (HUS) and Reiter's syndrome. The detection methods for shiga toxin needs to be rapid, accurate, reliable and must be extensively evaluated under field conditions. The aim of this study was to develop rapid, sensitive and specific detection method for Stx. Methods: : Mice and rabbits were immunized with purified recombinant Shiga toxin B (rStxB). Using these antibodies dot ELISA, sandwich ELISA and flow through assay were developed. Results: : The high-titre antibodies specifically reacted with purified rStxB. Dot-ELISA, sandwich ELISA and flow-through assay were developed and standardized that could detect StxB with limit of detection (LOD) of 9.75, 9.7 ng/ml and 0.46 µg/cassette, respectively. Interpretation & conclusions: : The rStxB was used to produce antibodies to avoid handling of pathogen. The Flow through assay 'developed was specific, rapid and field amenable.

Prediction of Shigellosis outcomes in Israel using machine learning classifiers.

Shigellosis causes significant morbidity and mortality in developing and developed countries, mostly among infants and young children. The World Health Organization estimates that more than one million people die from Shigellosis every year. In order to evaluate trends in Shigellosis in Israel in the years 2002-2015, we analysed national notifiable disease reporting data. Shigella sonnei was the most commonly identified Shigella species in Israel. Hospitalisation rates due to Shigella flexenri were higher in comparison with other Shigella species. Shigella morbidity was higher among infants and young children (age 0-5 years old). Incidence of Shigella species differed among various ethnic groups, with significantly high rates of S. flexenri among Muslims, in comparison with Jews, Druze and Christians. In order to improve the current Shigellosis clinical diagnosis, we developed machine learning algorithms to predict the Shigella species and whether a patient will be hospitalised or not, based on available demographic and clinical data. The algorithms' performances yielded an accuracy of 93.2% (Shigella species) and 94.9% (hospitalisation) and may consequently improve the diagnosis and treatment of the disease.