Development and application of a bacteriophage cocktail for Shigella flexneri biofilm inhibition on the stainless steel surface.

Food contamination and biofilm formation by Shigella in food processing facilities are major causes of acute gastrointestinal infection and mortality in humans. Bacteriophages (phages) are promising alternatives to antibiotics in controlling plankton and biofilms in food matrices. This study isolated two novel phages, S2_01 and S2_02, with lytic activity against various Shigella spp. From sewage samples. Transmission electron microscopy revealed that phages S2_01 and S2_02 belonged to the Caudovirales order. On characterizing their lytic ability, phage S2_01 initially exhibited relatively weak antibacterial activity, while phage S2_02 initially displayed rapid antibacterial activity after phage application. A combination of these phages in a 1:9 ratio was selected, as it has been suggested to elicit the most rapid and sustained lysis ability for up to 24 h. It demonstrated lytic activity against various foodborne pathogens, including six Shigella spp. The phage cocktail exhibited biofilm inhibition and disruption abilities of approximately 79.29% and 42.55%, respectively, after 24 h in a 96-well microplate. In addition, inhibition (up to 23.42%) and disruption (up to 19.89%) abilities were also observed on stainless steel surfaces, and plankton growth was also significantly suppressed. Therefore, the phage cocktail formulated in this study displays great potential as a biological control agent in improving food safety against biofilms and plankton.

Impact of eugenol on biofilm development in Shigella flexneri 1457: a plant terpenoid based-approach to inhibit food-borne pathogen.

Shigella flexneri is a gram-negative bacterium responsible for shigellosis and bacterial dysentery. Despite using various synthetic antimicrobial agents and antibiotics, their efficacy is limited, prompting concerns over antibiotic resistance and associated health risks. This study investigated eugenol, a polyphenol with inherent antioxidant and antibacterial properties, as a potential alternative treatment. We aimed to evaluate eugenol's antibacterial effects and mechanisms of action against S. flexneri and its impact on biofilm formation. We observed significant growth suppression of S. flexneri with eugenol concentrations of 8-10 mM (98.29%). Quantitative analysis using the Crystal Violet assay demonstrated a marked reduction in biofilm formation at 10 mM (97.01 %). Assessment of Cell Viability and morphology via Fluorescence-Activated Cell Sorting and Scanning Electron Microscopy confirmed these findings. Additionally, qPCR analysis revealed the downregulation of key genes responsible for adhesion (yebL), quorum sensing (rcsC, sdiA), and EPS production (s0482) associated with bacterial growth and biofilm formation. The present study suggests eugenol could offer a promising alternative to conventional antibiotics for treating shigellosis caused by S. flexneri.

Insights into the antibacterial effectiveness of linalool against Shigella flexneri on pork surface: Changes in bacterial growth and pork quality.

Shigella flexneri has the ability to contaminate pork and cause foodborne diseases. This study aimed to examine the effectiveness of linalool (a natural preservative) against S. flexneri and explore its potential application in contaminated pork. The results showed that linalool was capable of damaging the cell membrane and binding to the DNA of S. flexneri, and inhibiting biofilm formation and disrupting mature biofilms. The antibacterial effectiveness of linalool on the surface of pork was further demonstrated by analyzing the physicochemical properties of the pork (i.e., weight loss rate, pH value, color index, and TVB-N value) and its protein profiles. Linalool did not completely kill S. flexneri in pork at minimum bactericidal concentration (MBC) concentration and its antibacterial effect of linalool was stronger during the initial stage of storage. During storage, linalool influenced the abundance of specific proteins in the pork, particularly those involved in pathways related to fat metabolism. These findings offer novel insights into the antibacterial efficacy of linalool and its underlying mechanism in pork.

SRL pathogenicity island contributes to the metabolism of D-aspartate via an aspartate racemase in Shigella flexneri YSH6000.

In recent years, multidrug resistance of Shigella strains associated with genetic elements like pathogenicity islands, have become a public health problem. The Shigella resistance locus pathogenicity island (SRL PAI) of S. flexneri 2a harbors a 16Kbp region that contributes to the multidrug resistance phenotype. However, there is not much information about other functions such as metabolic, physiologic or ecological ones. For that, wild type S. flexneri YSH6000 strain, and its spontaneous SRL PAI mutant, 1363, were used to study the contribution of the island in different growth conditions. Interestingly, when both strains were compared by the Phenotype Microarrays, the ability to metabolize D-aspartic acid as a carbon source was detected in the wild type strain but not in the mutant. When D-aspartate was added to minimal medium with other carbon sources such as mannose or mannitol, the SRL PAI-positive strain was able to metabolize it, while the SRL PAI-negative strain did not. In order to identify the genetic elements responsible for this phenotype, a bioinformatic analysis was performed and two genes belonging to SRL PAI were found: orf8, coding for a putative aspartate racemase, and orf9, coding for a transporter. Thus, it was possible to measure, by an indirect analysis of racemization activity in minimal medium supplemented only with D-aspartate, that YSH6000 strain was able to transform the D-form into L-, while the mutant was impaired to do it. When the orf8-orf9 region from SRL island was transformed into S. flexneri and S. sonnei SRL PAI-negative strains, the phenotype was restored. Although, when single genes were cloned into plasmids, no complementation was observed. Our results strongly suggest that the aspartate racemase and the transporter encoded in the SRL pathogenicity island are important for bacterial survival in environments rich in D-aspartate.

Shigella flexneri Adherence Factor Expression in In Vivo-Like Conditions.

The Shigella species are Gram-negative, facultative intracellular pathogens that invade the colonic epithelium and cause significant diarrheal disease. Despite extensive research on the pathogen, a comprehensive understanding of how Shigella initiates contact with epithelial cells remains unknown. Shigella maintains many of the same Escherichia coli adherence gene operons; however, at least one critical gene component in each operon is currently annotated as a pseudogene in reference genomes. These annotations, coupled with a lack of structures upon microscopic analysis following growth in laboratory media, have led the field to hypothesize that Shigella is unable to produce fimbriae or other traditional adherence factors. Nevertheless, our previous analyses have demonstrated that a combination of bile salts and glucose induces both biofilm formation and adherence to colonic epithelial cells. The goal of this study was to perform transcriptomic and genetic analyses to demonstrate that adherence gene operons in Shigella flexneri strain 2457T are functional, despite the gene annotations. Our results demonstrate that at least three structural genes facilitate S. flexneri 2457T adherence for epithelial cell contact and biofilm formation. Furthermore, our results demonstrate that host factors, namely, glucose and bile salts at their physiological concentrations in the small intestine, offer key environmental stimuli required for adherence factor expression in S. flexneri This research may have a significant impact on Shigella vaccine development and further highlights the importance of utilizing in vivo-like conditions to study bacterial pathogenesis.IMPORTANCE Bacterial pathogens have evolved to regulate virulence gene expression at critical points in the colonization and infection processes to successfully cause disease. The Shigella species infect the epithelial cells lining the colon to result in millions of cases of diarrhea and a significant global health burden. As antibiotic resistance rates increase, understanding the mechanisms of infection is vital to ensure successful vaccine development. Despite significant gains in our understanding of Shigella infection, it remains unknown how the bacteria initiate contact with the colonic epithelium. Most pathogens harbor multiple adherence factors to facilitate this process, but Shigella was thought to have lost the ability to produce these factors. Interestingly, we have identified conditions that mimic some features of gastrointestinal transit and that enable Shigella to express adherence structural genes. This work highlights aspects of genetic regulation for Shigella adherence factors and may have a significant impact on future vaccine development.

A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria.

BACKGROUND: Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Therefore, rapid, accurate and low-cost methods represent valuable tools in determining bacterial ability to persist and proliferate within eukaryotic cells. RESULTS: Herein, we present the first application of the immunofluorescence In-Cell Western (ICW) assay aimed at quantifying intracellular bacteria in in vitro infection models. The performance of this new approach was evaluated in cell culture infection models using three microorganisms with different lifestyles. Two facultative intracellular bacteria, the fast-growing Shigella flexneri and a persistent strain of Escherichia coli, as well as the obligate intracellular bacterium Chlamydia trachomatis were chosen as bacterial models. The ICW assay was performed in parallel with conventional quantification methods, i.e. colony forming units (CFUs) and inclusion forming units (IFUs). The fluorescence signal intensity values from the ICW assay were highly correlated to CFU/IFUs counting and showed coefficients of determination (R2), ranging from 0,92 to 0,99. CONCLUSIONS: The ICW assay offers several advantages including sensitivity, reproducibility, high speed, operator-independent data acquisition and overtime stability of fluorescence signals. All these features, together with the simplicity in performance, make this assay particularly suitable for high-throughput screening and diagnostic approaches.

Enhanced Production of Shigella flexneri Polysaccharide from a Newly Devised Silicate Method as a Potential Vaccine Conjugate.

Conventional glycoconjugates are prepared with polysaccharides (PS) isolated from bacterial sources by fermentation technique. This approach has some major challenges like lower yield of PS, impurities and usage of hazardous chemicals. Reports on efficient and enhanced production of PS from Shigella flexneri is meager in literature. Hence, in the current study, three different types of media namely Yeast extract medium, Shigella sonnei-defined medium and synthetic medium were utilized for the culture of S. flexneri. Among the selected media it was recognized that the culture of S. flexneri harvested in synthetic media produced significant quantity of PS in less time when compared to the other two media. Different purification techniques such as phenol chloroform extraction, acid precipitation, detergent method, chromatographic purification and a novel silicate method were carried out to refine the harvested PS from impurities. It was observed that large impurities such as bacterial protein, debris and media components were eliminated significantly by using chromatographic and silicate methods. The final yield of purified PS was approximately 20-35% higher in silicate method which is reported for the first time in this study for purification of PS. Further, the characterization of the purified PS was done using high-performance liquid chromatography and high-performance anion-exchange chromatography with pulsed amperometric detection. Hence, the robust process developed in the present study using synthetic media and chromatographic filtration technique along with the novel silicate treatment produced significant quantities of PS from S. flexneri in reduced cost and time, which could be further conjugated to a suitable carrier to generate a potential Shigella conjugate antigen.

In vitro antimicrobial effects of Hypoxis hemerocallidea against six pathogens with dermatological relevance and its phytochemical characterization and cytotoxicity evaluation.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypoxis hemerocallidea (commonly known as African Potato) is popular in African traditional medicine. It is used in the management of diverse ailments including burns, wounds and skin-related diseases. AIM OF THE STUDY: The current study investigated the antimicrobial effects of Hypoxis hemerocallidea against six microorganisms associated with skin diseases. In addition, the antioxidant activity, phytochemical profiles and cytotoxicity of the bulb extracts were evaluated. MATERIALS AND METHODS: The antimicrobial activity of 50% methanol (MeOH) and petroleum ether (PE) extracts of Hypoxis hemerocallidea bulbs was tested against two bacterial and four fungal strains implicated in causing opportunistic skin-related diseases. Antioxidant potential of the extract was investigated via the 2,2-diphenyl-1-picryhydrazyl (DPPH) free radical scavenging assay and ß-carotene linoleic acid model. Phytochemical profiling of the 50% MeOH extract of Hypoxis hemerocallidea was done using spectrophotometric assay and Gas Chromatography-Mass Spectrometry (GC-MS). The extracts were also evaluated for cytotoxicity against African green monkey Vero kidney cell lines based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. RESULTS: Both 50% MeOH and PE extracts showed considerable inhibitory effects against all six microorganisms. The extracts were potent against Shigella flexneri and Trichophyton tonsurans with minimum inhibitory concentration (MIC) values less than 1 mg/ml. However, there was relatively poor antifungal activity against the other fungal strains. In the DPPH assay, the MeOH extract of the bulb had an EC50 of 29.8 µg/ml while 76.91% antioxidant activity was observed in the ß-carotene-linoleic acid model. The extract contained total phenolics (41 mg GAE/g) and flavonoids (10 mg CE/g). The GC-MS analysis of Hypoxis hemerocallidea bulb revealed 29 and 160 bioactive compounds for 50% MeOH and PE extracts, respectively. Based on the cytotoxicity, Hypoxis hemerocallidea had LC50 value of 210.9 ±â€¯18.4 and 95.5 ±â€¯13.3 µg/ml for PE and MeOH extracts, respectively. CONCLUSIONS: The bulb extracts of Hypoxis hemerocallidea exhibited good antimicrobial and antioxidant activities, which could be attributed to the presence of phenolics, flavonoids and the other bioactive compounds identified through GC-MS, making it a potentially effective cosmetic plant. These findings also account for the multi-pharmacological use of Hypoxis hemerocallidea in traditional medicine, especially related to skin diseases. The plant extracts can be considered as safe based on their LC50 values (< 20 µg/ml). However, other form of cytotoxicity studies need to be carried out on Hypoxis hemerocallidea, as well as in vivo tests, to confirm its safety and efficacy as a treatment for skin-related diseases.

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.

A murine model of diarrhea, growth impairment and metabolic disturbances with Shigella flexneri infection and the role of zinc deficiency.

Shigella is one of the major enteric pathogens worldwide. We present a murine model of S. flexneri infection and investigate the role of zinc deficiency (ZD). C57BL/6 mice fed either standard chow (HC) or ZD diets were pretreated with an antibiotic cocktail and received S. flexneri strain 2457T orally. Antibiotic pre-treated ZD mice showed higher S. flexneri colonization than non-treated mice. ZD mice showed persistent colonization for at least 50 days post-infection (pi). S. flexneri-infected mice showed significant weight loss, diarrhea and increased levels of fecal MPO and LCN in both HC and ZD fed mice. S. flexneri preferentially colonized the colon, caused epithelial disruption and inflammatory cell infiltrate, and promoted cytokine production which correlated with weight loss and histopathological changes. Infection with S. flexneri ΔmxiG (critical for type 3 secretion system) did not cause weight loss or diarrhea, and had decreased stool shedding duration and tissue burden. Several biochemical changes related to energy, inflammation and gut-microbial metabolism were observed. Zinc supplementation increased weight gains and reduced intestinal inflammation and stool shedding in ZD infected mice. In conclusion, young antibiotic-treated mice provide a new model of oral S. flexneri infection, with ZD promoting prolonged infection outcomes.

Evaluating Shigella flexneri Pathogenesis in the Human Enteroid Model.

The enteric pathogen Shigella is one of the leading causes of moderate-to-severe diarrhea and death in young children in developing countries. Transformed cell lines and animal models have been widely used to study Shigella pathogenesis. In addition to altered physiology, transformed cell lines are composed of a single cell type that does not sufficiently represent the complex multicellular environment of the human colon. Most available animal models do not accurately mimic human disease. The human intestinal enteroid model, derived from LGR5+ stem cell-containing intestinal crypts from healthy subjects, represents a technological leap in human gastrointestinal system modeling and provides a more physiologically relevant system that includes multiple cell types and features of the human intestine. We established the utility of this model for studying basic aspects of Shigella pathogenesis and host responses. In this study, we show that Shigellaflexneri is capable of infecting and replicating intracellularly in human enteroids derived from different segments of the intestine. Apical invasion by S. flexneri is very limited but increases ∼10-fold when enteroids are differentiated to include M cells. Invasion via the basolateral surface was at least 2-log10 units more efficient than apical infection. Increased secretion of interleukin-8 and higher expression levels of the mucin glycoprotein Muc2 were observed in the enteroids following S. flexneri infection. The human enteroid model promises to bridge some of the gaps between traditional cell culture, animal models, and human infection.

Shigella effector IpaH4.5 targets 19S regulatory particle subunit RPN13 in the 26S proteasome to dampen cytotoxic T lymphocyte activation.

Subversion of antigen-specific immune responses by intracellular pathogens is pivotal for successful colonisation. Bacterial pathogens, including Shigella, deliver effectors into host cells via the type III secretion system (T3SS) in order to manipulate host innate and adaptive immune responses, thereby promoting infection. However, the strategy for subverting antigen-specific immunity is not well understood. Here, we show that Shigella flexneri invasion plasmid antigen H (IpaH) 4.5, a member of the E3 ubiquitin ligase effector family, targets the proteasome regulatory particle non-ATPase 13 (RPN13) and induces its degradation via the ubiquitin-proteasome system (UPS). IpaH4.5-mediated RPN13 degradation causes dysfunction of the 19S regulatory particle (RP) in the 26S proteasome, inhibiting guidance of ubiquitinated proteins to the proteolytically active 20S core particle (CP) of 26S proteasome and thereby suppressing proteasome-catalysed peptide splicing. This, in turn, reduces antigen cross-presentation to CD8+ T cells via major histocompatibility complex (MHC) class I in vitro. In RPN13 knockout mouse embryonic fibroblasts (MEFs), loss of RPN13 suppressed CD8+ T cell priming during Shigella infection. Our results uncover the unique tactics employed by Shigella to dampen the antigen-specific cytotoxic T lymphocyte (CTL) response.

Shutting Down Shigella Secretion: Characterizing Small Molecule Type Three Secretion System ATPase Inhibitors.

Many important human pathogens rely on one or more type three secretion systems (T3SSs) to inject bacterial effector proteins directly into the host cell cytoplasm. Secretion of protein through the needlelike type three secretion apparatus (T3SA) is essential for pathogen virulence and relies on a highly conserved ATPase at the base of the apparatus, making it an attractive target for anti-infective therapeutics. Here, we leveraged the ability to purify an active oligomeric Shigella T3SS ATPase to provide kinetic analyses of three T3SS ATPase inhibitors of Spa47. In agreement with in silico docking simulations, the inhibitors displayed noncompetitive inhibition profiles and efficiently reduced Spa47 ATPase activity with IC50s as low as 52 ± 3 µM. Two of the inhibitors functioned well in vivo, nearly abolishing effector protein secretion without significantly affecting the Shigella growth phenotype or HeLa cell viability. Furthermore, characterization of Spa47 complexes in vitro and Shigella T3SA formation in vivo showed that the inhibitors do not function through disruption of Spa47 oligomers or by preventing T3SA formation. Together, these findings suggest that inhibitors targeting Spa47 may be an effective means of combating Shigella infection by shutting down type three secretion without preventing presentation of the highly antigenic T3SA tip proteins that aid in clearing the infection and developing pan- Shigella immunological memory. In summary, this is the first report of Shigella T3SS ATPase inhibitors and one of only a small number of studies characterizing T3SS ATPase inhibition in general. The work presented here provides much-needed insight into T3SS ATPase inhibition mechanisms and provides a strong platform for developing and evaluating non-antibiotic therapeutics targeting Spa47 and other T3SS ATPases.

Spatial, Temporal, and Functional Assessment of LC3-Dependent Autophagy in Shigella flexneri Dissemination.

Shigella flexneri disseminates within the colonic mucosa by displaying actin-based motility in the cytosol of epithelial cells. Motile bacteria form membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the bacteria escape, thereby achieving cell-to-cell spread. During dissemination, S. flexneri is targeted by LC3-dependent autophagy, a host cell defense mechanism against intracellular pathogens. The S. flexneri type III secretion system effector protein IcsB was initially proposed to counteract the recruitment of the LC3-dependent autophagy machinery to cytosolic bacteria. However, a recent study proposed that LC3 was recruited to bacteria in DMVs formed during cell-to-cell spread. To resolve the controversy and clarify the role of autophagy in S. flexneri infection, we tracked dissemination using live confocal microscopy and determined the spatial and temporal recruitment of LC3 to bacteria. This approach demonstrated that (i) LC3 was exclusively recruited to wild-type or icsB bacteria located in DMVs and (ii) the icsB mutant was defective in cell-to-cell spread due to failure to escape LC3-positive as well as LC3-negative DMVs. Failure of S. flexneri to escape DMVs correlated with late LC3 recruitment, suggesting that LC3 recruitment is the consequence and not the cause of DMV escape failure. Inhibition of autophagy had no positive impact on the spreading of wild-type or icsB mutant bacteria. Our results unambiguously demonstrate that IcsB is required for DMV escape during cell-to-cell spread, regardless of LC3 recruitment, and do not support the previously proposed notion that autophagy counters S. flexneri dissemination.

The role of lipids in host-pathogen interactions.

Innate immunity relies on the effective recognition and elimination of pathogenic microorganisms. This entails sequestration of pathogens into phagosomes that promptly acquire microbicidal and degradative properties. This complex series of events, which involve cytoskeletal reorganization, membrane remodeling and the activation of multiple enzymes, is orchestrated by lipid signaling. To overcome this immune response, intracellular pathogens acquired mechanisms to subvert phosphoinositide-mediated signaling and use host lipids, notably cholesterol, as nutrients. We present brief overviews of the role of phosphoinositides in phagosome formation and maturation as well as of cholesterol handling by host cells, and selected Salmonella, Shigella, Chlamydia and Mycobacterium tuberculosis to exemplify the mechanisms whereby intracellular pathogens co-opt lipid metabolism in host cells. © 2018 IUBMB Life, 70(5):384-392, 2018.

Shigella hijacks the glomulin-cIAPs-inflammasome axis to promote inflammation.

Shigella deploys a unique mechanism to manipulate macrophage pyroptosis by delivering the IpaH7.8 E3 ubiquitin ligase via its type III secretion system. IpaH7.8 ubiquitinates glomulin (GLMN) and elicits its degradation, thereby inducing inflammasome activation and pyroptotic cell death of macrophages. Here, we show that GLMN specifically binds cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1 and cIAP2), members of the inhibitor of apoptosis (IAP) family of RING-E3 ligases, which results in reduced E3 ligase activity, and consequently inflammasome-mediated death of macrophages. Importantly, reducing the levels of GLMN in macrophages via IpaH7.8, or siRNA-mediated knockdown, enhances inflammasome activation in response to infection by Shigella, Salmonella, or Pseudomonas, stimulation with NLRP3 inflammasome activators (including SiO2, alum, or MSU), or stimulation of the AIM2 inflammasome by poly dA:dT GLMN binds specifically to the RING domain of both cIAPs, which inhibits their self-ubiquitination activity. These findings suggest that GLMN is a negative regulator of cIAP-mediated inflammasome activation, and highlight a unique Shigella stratagem to kill macrophages, promoting severe inflammation.

Adaptations in the physiological heterogeneity and viability of Shigella dysenteriae, Shigella flexneri and Salmonella typhimurium, after exposure to simulated gastric acid fluid.

Stomach acidity is an important barrier of the human body to protect itself from microbial pathogens entering the small intestine and causing infection. This study examined the survival adaptations of non-acid adapted diarrheal Shigella and Salmonella strains in an environment mimicking the human stomach. The bacterial responses to the challenge of acidic simulated gastric fluid were studied using flow cytometry physiological heterogeneity, membrane integrity and survival (culturability) respectively. Flow cytometry showed that bacterial cells, when exposed to gastric fluid, transformed distinctly, into physiologically heterogeneous sub-populations: intact, stressed and damaged cells, when stained with propidium iodide and thiazole orange. Shigella and Salmonella cells became membrane compromised during initial acid shock (0-30 min), and 80% of these cells shifted to the stressed state throughout gastric fluid exposure. Approximately 10-30% of bacterial strains remained culturable after 60 min of gastric fluid exposure at pH 2.5-4.5, with the percentage increasing with an inoculum size of 102 CFU/ml. This ability of non-acid adapted Shigella and Salmonella sp. to adapt and survive low pH gastric fluid, even though the bacterial numbers decreased or changed to a stressed state, further supports the possible risk of infection when consumed.

Ubiquitination and degradation of GBPs by a Shigella effector to suppress host defence.

Interferon-inducible guanylate-binding proteins (GBPs) mediate cell-autonomous antimicrobial defences. Shigella flexneri, a Gram-negative cytoplasmic free-living bacterium that causes bacillary dysentery, encodes a repertoire of highly similar type III secretion system effectors called invasion plasmid antigen Hs (IpaHs). IpaHs represent a large family of bacterial ubiquitin-ligases, but their function is poorly understood. Here we show that S. flexneri infection induces rapid proteasomal degradation of human guanylate binding protein-1 (hGBP1). We performed a transposon screen to identify a mutation in the S. flexneri gene ipaH9.8 that prevented hGBP1 degradation. IpaH9.8 targets hGBP1 for degradation via Lys48-linked ubiquitination. IpaH9.8 targets multiple GBPs in the cytoplasm independently of their nucleotide-bound states and their differential function in antibacterial defence, promoting S. flexneri replication and resulting in the death of infected mice. In the absence of IpaH9.8, or when binding of GBPs to IpaH9.8 was disrupted, GBPs such as hGBP1 and mouse GBP2 (mGBP2) translocated to intracellular S. flexneri and inhibited bacterial replication. Like wild-type mice, mutant mice deficient in GBP1-3, 5 and 7 succumbed to S. flexneri infection, but unlike wild-type mice, mice deficient in these GBPs were also susceptible to S. flexneri lacking ipaH9.8. The mode of IpaH9.8 action highlights the functional importance of GBPs in antibacterial defences. IpaH9.8 and S. flexneri provide a unique system for dissecting GBP-mediated immunity.

Listeria monocytogenes and Shigella flexneri Activate the NLRP1B Inflammasome.

Activation of the innate immune receptor NLRP1B leads to the formation of an inflammasome, which induces autoproteolytic processing of pro-caspase-1, and ultimately to the release of inflammatory cytokines and to the execution of pyroptosis. One of the signals to which NLRP1B responds is metabolic stress that occurs in cells deprived of glucose or treated with metabolic inhibitors. NLRP1B might therefore sense microbial infection, as intracellular pathogens such as Listeria monocytogenes and Shigella flexneri cause metabolic stress as a result of nutrient scavenging and host cell damage. Here we addressed whether these pathogens activate the NLRP1B inflammasome. We found that Listeria infection activated the NLRP1B inflammasome in a reconstituted fibroblast model. Activation of NLRP1B by Listeria was diminished in an NLRP1B mutant shown previously to be defective at detecting energy stress and was dependent on the expression of listeriolysin O (LLO), a protein required for vacuolar escape. Infections of either Listeria or Shigella activated NLRP1B in the RAW264.7 murine macrophage line, which expresses endogenous NLRP1B. We conclude that NLRP1B senses cellular infection by distinct invasive pathogens.

Inhibition of Shigella sonnei and Shigella flexneri in Hummus Using Citric Acid and Garlic Extract.

Hummus (chickpea dip) is a ready-to-eat product that may pose a significant risk to human if pathogens are present. Several organisms including Shigella spp. have been isolated from hummus. However, studies on the survival and inhibition of Shigella spp. in food are scarce. This study investigated the growth pattern of Sh. sonnei and Sh. flexneri in hummus at different temperatures (4, 10, and 24 °C). Additionally, the inhibitory activity of different concentrations of citric acid (CA) (0.5%, 1.0%, and 2.0%) and garlic extract (GE) (1.0%, 2.0%, and 3.0%) against Sh. sonnei and Sh. flexneri inoculated into hummus and stored at 4 and 10 °C was investigated. Both Shigella spp. survived well at 4 °C, while both grew to >7.0 log10 after 4 d at 10 °C or 1 d at 24 °C. At 4 °C, CA at 0.5% and 1.0% resulted in a slight reduction in the count (approximately 1.0 log10 ); a complete elimination of Sh. sonnei was attained by using 2.0% CA. However, approximately 3.0 log10 reduction in Sh. sonnei was obtained at 10 °C. For Sh. flexneri, CA at 0.5% and 1.0% resulted in a bacteriostatic inhibition. GE at 1.0% and 2.0% resulted in approximately 1.0 to 2.0 log10 reduction in Sh. sonnei count at 4 °C, while at 3.0% GE, approximately 4.0 and 3.0 log10 reductions were obtained at 4 and 10 °C, respectively. In comparison, the 2.0% and 3.0% GE resulted in a bacteriostatic effect against Sh. flexneri at 4 and 10 °C.