A Tale about Shigella: Evolution, Plasmid, and Virulence.

Shigella spp. cause hundreds of millions of intestinal infections each year. They target the mucosa of the human colon and are an important model of intracellular bacterial pathogenesis. Shigella is a pathovar of Escherichia coli that is characterized by the presence of a large invasion plasmid, pINV, which encodes the characteristic type III secretion system and icsA used for cytosol invasion and cell-to-cell spread, respectively. First, we review recent advances in the genetic aspects of Shigella, shedding light on its evolutionary history within the E. coli lineage and its relationship to the acquisition of pINV. We then discuss recent insights into the processes that allow for the maintenance of pINV. Finally, we describe the role of the transcription activators VirF, VirB, and MxiE in the major virulence gene regulatory cascades that control the expression of the type III secretion system and icsA. This provides an opportunity to examine the interplay between these pINV-encoded transcriptional activators and numerous chromosome-encoded factors that modulate their activity. Finally, we discuss novel chromosomal genes icaR, icaT, and yccE that are regulated by MxiE. This review emphasizes the notion that Shigella and E. coli have walked the fine line between commensalism and pathogenesis for much of their history.

The T3SS of Shigella: Expression, Structure, Function, and Role in Vacuole Escape.

Shigella spp. are one of the leading causes of infectious diarrheal diseases. They are Escherichia coli pathovars that are characterized by the harboring of a large plasmid that encodes most virulence genes, including a type III secretion system (T3SS). The archetypal element of the T3SS is the injectisome, a syringe-like nanomachine composed of approximately 20 proteins, spanning both bacterial membranes and the cell wall, and topped with a needle. Upon contact of the tip of the needle with the plasma membrane, the injectisome secretes its protein substrates into host cells. Some of these substrates act as translocators or effectors whose functions are key to the invasion of the cytosol and the cell-to-cell spread characterizing the lifestyle of Shigella spp. Here, we review the structure, assembly, function, and methods to measure the activity of the injectisome with a focus on Shigella, but complemented with data from other T3SS if required. We also present the regulatory cascade that controls the expression of T3SS genes in Shigella. Finally, we describe the function of translocators and effectors during cell-to-cell spread, particularly during escape from the vacuole, a key element of Shigella's pathogenesis that has yet to reveal all of its secrets.

Natural products and polysorbates: Potential Inhibitors of biofilm formation in Pseudomonas aeruginosa.

INTRODUCTION: With all the challenges super bugs are imposing, biofilm formation opens the door against various more complicated challenges. Such issue may be highlighted with the ability of the latter to render the antibiotics hardly accessible to bacterial cells and sheds the light on the importance of finding antibiofilm formers. Therefore, we assessed the inhibitory effect of natural product extracts (ginger, wild blueberry) and polysorbates (PS20, PS80) on biofilm formation at the molecular level. METHODOLOGY: Growth inhibition assay was performed to test the effect of ginger (Zingiber Officinale), wild blueberry (Vaccinium Angustifolium), and polysorbates on Pseudomonas aeruginosa (PAN14) growth. Transcription levels of biofilm exopolysaccharides encoding genes (ndvB, pelC, algC) and quorum sensing genes (lasI, lasR, rhlI, rhlR) for LasI/LasR and RhlI/ RhlR systems were evaluated by RT qPCR. RESULTS: The polysorbates and the extracts of both ginger and wild blueberry had no effect on the growth of P. aeruginosa. Biofilms' examination has unraveled the effectiveness of treatments used in reducing its formation. Moreover, a significant reduction in the expression of all genes tested for biofilm exopolysaccharides and its quorum sensing system was observed. CONCLUSION: The decrease in the relative gene expression of the exopolysaccharides and quorum sensing encoding genes sheds the light on the mechanism of action of ginger and wild blueberry's constituents as well as polysorbates 20 and 80 on P. aeruginosa biofilm formation. Future studies need to assess the antibiofilm effect of each fraction of herbal extracts separately.

PulseNet Lebanon: An Overview of Its Activities, Outbreak Investigations, and Challenges.

Background: Foodborne diseases are still a major health issue in Lebanon, although some steps have been taken forward in food safety. To this purpose, PulseNet Lebanon, a foodborne diseases tracking network, was established in 2009, through the collaboration between the Ministry of Public Health (MoPH) and the American University of Beirut (AUB). Materials and Methods: Three papers published regarding the PulseNet project were summarized. Initially, clinical and food samples, collected within the surveillance network scope, were identified by using the respective API for Salmonella and Listeria spp. Salmonella spp. were further serotyped by using the Kauffman and White method. Campylobacter spp. were determined by the 16 S rRNA sequencing method. Antimicrobial susceptibility to a number of antibiotics was determined by using the disk diffusion method for Samonella and Campylobacter spp. Genomic diversity was determined by using pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD). Results: Results indicated that 290 clinical and 49 food isolates were identified as Salmonella. Serotyping revealed the prevalence of ten and seven serotypes in the clinical and food samples, respectively. Fifty-one isolates from chicken ceca and carcass were identified to be Campylobacter spp. Fifty-nine samples were identified to be Listeria monocytogenes. Antimicrobial susceptibility testing revealed a wide range of resistance among the different samples. PFGE showed a variation in pulsotypes among the Salmonella serotypes. PFGE also linked certain outbreaks to their food sources. This method also demonstrated 13 subtypes with 100% similarity among the L. monocytogenes isolates. Finally, the Camplyobcater spp. were grouped into nine clusters with a minimum similarity of 43.5% using RAPD. Conclusion: This summary of results shows the importance of implementing a "farm-to-fork" approach in the surveillance of foodborne disease outbreaks in Lebanon, allowing the detection of pathogens causing foodborne disease outbreaks in a timely fashion.

Epstein-Barr Virus DNA Enhances Diptericin Expression and Increases Hemocyte Numbers in Drosophila melanogaster via the Immune Deficiency Pathway.

Infection with the Epstein-Barr virus (EBV) is associated with several malignancies and autoimmune diseases in humans. The following EBV infection and establishment of latency, recurrences frequently occur resulting in potential viral DNA shedding, which may then trigger the activation of immune pathways. We have previously demonstrated that levels of the pro-inflammatory cytokine IL-17, which is associated with several autoimmune diseases, are increased in response to EBV DNA injection in mice. Whether other pro-inflammatory pathways are induced in EBV DNA pathobiology remains to be investigated. The complexity of mammalian immune systems presents a challenge to studying differential activities of their intricate immune pathways in response to a particular immune stimulus. In this study, we used Drosophila melanogaster to identify innate humoral and cellular immune pathways that are activated in response to EBV DNA. Injection of wild-type adult flies with EBV DNA induced the immune deficiency (IMD) pathway resulting in enhanced expression of the antimicrobial peptide diptericin. Furthermore, EBV DNA increased the number of hemocytes in flies. Conditional silencing of the IMD pathway decreased diptericin expression in addition to curbing of hemocyte proliferation in response to challenge with EBV DNA. Comparatively, upon injecting mice with EBV DNA, we detected enhanced expression of tumor necrosis factor-α (TNFα); this enhancement is rather comparable to IMD pathway activation in flies. This study hence indicates that D. melanogaster could possibly be utilized to identify immune mediators that may also play a role in the response to EBV DNA in higher systems.

Evaluating ginger extract, wild blueberry extract, and polysorbates (PS20, PS80) on Pseudomonas aeruginosa biofilm formation.

INTRODUCTION: Pseudomonas aeruginosa is a biofilm forming pathogen that challenges clinical and industrial settings. Many natural products and surfactants have been screened and valued for their anti-biofilm capacity. In this study we assessed the inhibitory effect and molecular mechanism of action of ginger extract (Zingiber officinale Rosc.), wild blueberry extract (Vaccinium angustifolium), and polysorbates (PS20/PS80) on biofilm formation. METHODOLOGY: Ginger and wild blueberry extractions were done using ethanol and distilled water, respectively. Hexane and methanol were used for extracts' liquid-liquid portioning. LC-HRMS was performed to obtain extract fractions. Efficacy of the crude extracts, fractions, and polysorbates was assessed on P. aeruginosa PAN14 growth and biofilm. Transcription levels of biofilm encoding genes ndvB, pelC, algC and quorum sensing genes lasI, lasR, rhlI, rhlR were evaluated by RT-qPCR. RESULTS: Extracts and polysorbates concentrations did not affect P. aeruginosa growth. Biofilm assay showed a reduction in biofilm when 5% ginger, 25% wild blueberry extracts, 0.2% PS20, and 0.25% PS80 were added. LC-HRMS analysis of ginger extract showed abundant gingerol in the hexane layer. Wild blueberry chromatograms showed various constituents differing between their peel and pulp, and pulp extracts. RT-qPCR showed decreased transcription levels of exopolysaccharide and quorum sensing genes with a 363.6 folds reduction in ndvB upon treatment with 25% wild blueberry peel and pulp extract. CONCLUSION: These results shed light on the mechanism of action of ginger and wild blueberry constituents as well as PS20/80 on P. aeruginosa biofilm formation. Future mouse model experiments are useful to test biofilm inhibition in-vivo.

Extracellular vesicles released by mesenchymal-like prostate carcinoma cells modulate EMT state of recipient epithelial-like carcinoma cells through regulation of AR signaling.

Extracellular vesicles released from cancer cells may play an important role in cancer progression by shuttling oncogenic information into recipient cells. However, our knowledge is still fragmentary and there remain numerous questions regarding the mechanisms at play and the functional consequences of these interactions. We have recently established a mesenchymal-like prostate cancer cell line (22Rv1/CR-1; Mes-PCa). In this study, we assessed the effects of the extracellular vesicles released by these cells on recipient androgen-dependent epithelial VCaP prostate cancer cells. Mes-PCa derived vesicles were found to promote mesenchymal features in the recipient epithelial-like prostate cancer cells. This transformation was accompanied by a modulation of androgen receptor signaling and activation of TGFß signaling pathway. Moreover, recipient cells acquiring mesenchymal traits displayed enhanced migratory and invasive features as well as increased resistance to the androgen receptor antagonist, enzalutamide. Our results suggest a previously unappreciated role for Mes-PCa secreted vesicles in cancer promotion by transferring cell-mediated signals and promoting phenotypic changes in recipient prostate cancer cells.

Genotypic and virulence characteristics of Listeria monocytogenes recovered from food items in Lebanon.

INTRODUCTION: Listeria monocytogenes is the agent of listeriosis, a life threatening foodborne disease for immunocompromised patients and pregnant women. This bacterium is not routinely screened for in Lebanon and there is lack of data about the prevalent strains and their potential pathogenicity. To that purpose, this study was undertaken to characterize L. monocytogenes from various food products, by assessing the in vitro biofilm forming ability, detecting their virulence potential, and characterizing them at the strain level. METHODOLOGY: Fifty-nine isolates were obtained from the Lebanese Agriculture Research Institute (LARI). They were collected in 2012-2013 from local and imported food products in the Lebanese market. Biofilm formation was measured using the Microtiter Plate Assay. PCR amplification was performed for three main virulence genes; hly, actA, and inlB. Pulsed field gel electrophoresis (PFGE) and BIONUMERICS analysis were carried out. RESULTS: Lebanese isolates from cheese and raw meat showed higher biofilm formation than imported and Lebanese seafood isolates. A total of 100% of the isolates were PCR positive for hly and actA genes and 98.3% for inlB gene. PFGE analysis demonstrated the prevalence of 13 different subtypes with 100% similarity. Detected subtypes were grouped into 6 clusters of 90% genomic similarity. Clustered subtypes were particular to the country of origin. CONCLUSION: This study highlights the presence of L. monocytogenes in the Lebanese food market with high pathogenic potential and stresses the importance of enhanced surveillance and the implementation of strict regulations on local and imported food. Future investigations may be conducted on a larger food selection.

Corrigendum: Escherichia coli Isolated from Urinary Tract Infections of Lebanese Patients between 2005 and 2012: Epidemiology and Profiles of Resistance.

[This corrects the article on p. 26 in vol. 2, PMID: 4415468.].