Shigella Infection Research Articles

Development of Shigella conjugate vaccines targeting Shigella flexneri 2a and S. flexneri 3a using a simple platform-approach conjugation by squaric acid chemistry

There is a need for vaccines effective against shigella infection in young children in resource-limited areas. Protective immunity against shigella infection targets the O-specific polysaccharide (OSP) component of lipopolysaccharide. Inducing immune responses to polysaccharides in young children can be problematic, but high level and durable responses can be induced by presenting polysaccharides conjugated to carrier proteins. An effective shigella vaccine will need to be multivalent, targeting the most common global species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. Here we report the development of shigella conjugate vaccines (SCV) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using squaric acid chemistry to result in single point sun-burst type display of OSP from carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. We confirmed structure and demonstrated that these conjugates were recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG responses, as well as rTTHc-specific IgG responses. Vaccination induced serotype-specific bactericidal antibody responses against S. flexneri, and vaccinated animals were protected against keratoconjunctivitis (Sereny test) and intraperitoneal challenge with virulent S. flexneri 2a and 3a, respectively. Our results support further development of this platform conjugation technology in the development of shigella conjugate vaccines for use in resource-limited settings.

The long view of vaccination to secure the future

The long view of vaccination to secure the future

Projecting the long-term economic benefits of reducing Shigella-attributable linear growth faltering with a potential vaccine: a modelling study

Linear growth is an important outcome of child development with implications for economic productivity. Enteric infections, particularly Shigella, have been linked to linear growth faltering (LGF). However, benefits from potential reductions in LGF are rarely included in economic analyses of enteric infections. We aimed to quantify the economic benefits of vaccination related to reduced Shigella-attributable disease and associated LGF compared with the net costs of a vaccine programme. In this benefit-cost analysis, we modelled productivity benefits in 102 low-income and middle-income countries that had recent stunting estimates available, at least one Shigella-attributable death annually, and available economic data, particularly on gross national income and growth rate projections. We modelled benefits strictly related to linear growth improvements and no other benefits associated with reducing diarrhoeal burden. The effect size in each country was calculated as shifts in height-for-age Z score (HAZ), representing population average changes for preventing Shigella-attributable less-severe diarrhoea and moderate-to-severe diarrhoea separately for children younger than 5 years. Benefits data were calculated per country and combined with estimated net costs of the vaccine programme in the form of benefit-cost ratios (BCRs); BCRs above parity, or $1 in benefits per $1 in costs (with a 10% margin representing borderline results: 1·10:1), were considered cost-beneficial. Countries were aggregated for analysis by WHO region, World Bank income category, and eligibility for support from Gavi, the Vaccine Alliance. In the base-case scenario, all regions exhibited cost-beneficial results, with the South-East Asia region and Gavi-eligible countries exhibiting the highest BCRs (21·67 for the South-East Asia region and 14·45 for Gavi-eligible countries), and the Eastern Mediterranean region yielding the lowest BCRs (2·90). All regions exhibited cost-beneficial results from vaccination, except in more conservative scenarios (eg, those assuming early retirement ages and higher discount rates). Our findings were sensitive to assumed returns for increased height, assumptions about vaccine efficacy against linear growth detriments, the anticipated shift in HAZ, and discount rate. Incorporating the productivity benefits of LGF reduction into existing cost-effectiveness estimates resulted in longer-term cost-savings in nearly all regions. LGF is a secondary outcome of Shigella infection and reduction in LGF is not often quantified as a health or economic benefit of vaccination. However, even under conservative assumptions, a Shigella vaccine only moderately effective against LGF could pay for itself from productivity gains alone in some regions. We recommend that LGF be considered in future models assessing the economic and health impacts of interventions preventing enteric infections. Further research is needed on vaccine efficacy against LGF to inform such models. Bill & Melinda Gates Foundation, Wellcome Trust.

Functional assays to evaluate antibody-mediated responses against Shigella: a review.

Shigella is a major global pathogen and the etiological agent of shigellosis, a diarrheal disease that primarily affects low- and middle-income countries. Shigellosis is characterized by a complex, multistep pathogenesis during which bacteria use multiple invasion proteins to manipulate and invade the intestinal epithelium. Antibodies, especially against the O-antigen and some invasion proteins, play a protective role as titres against specific antigens inversely correlate with disease severity; however, the context of antibody action during pathogenesis remains to be elucidated, especially with Shigella being mostly an intracellular pathogen. In the absence of a correlate of protection, functional assays rebuilding salient moments of Shigella pathogenesis can improve our understanding of the role of protective antibodies in blocking infection and disease. In vitro assays are important tools to build correlates of protection. Only recently animal models to recapitulate human pathogenesis, often not in full, have been established. This review aims to discuss in vitro assays to evaluate the functionality of anti-Shigella antibodies in polyclonal sera in light of the multistep and multifaced Shigella infection process. Indeed, measurement of antibody level alone may limit the evaluation of full vaccine potential. Serum bactericidal assay (SBA), and other functional assays such as opsonophagocytic killing assays (OPKA), and adhesion/invasion inhibition assays (AIA), are instead physiologically relevant and may provide important information regarding the role played by these effector mechanisms in protective immunity. Ultimately, the review aims at providing scientists in the field with new points of view regarding the significance of functional assays of choice which may be more representative of immune-mediated protection mechanisms.

Role of the MDR Efflux Pump AcrAB in Epithelial Cell Invasion by Shigella flexneri.

The tripartite complex AcrAB-TolC is the major RND pump in Escherichia coli and other Enterobacteriaceae, including Shigella, the etiological agent of bacillary dysentery. In addition to conferring resistance to many classes of antibiotics, AcrAB plays a role in the pathogenesis and virulence of several bacterial pathogens. Here, we report data demonstrating that AcrAB specifically contributes to Shigella flexneri invasion of epithelial cells. We found that deletion of both acrA and acrB genes causes reduced survival of S. flexneri M90T strain within Caco-2 epithelial cells and prevents cell-to-cell spread of the bacteria. Infections with single deletion mutant strains indicate that both AcrA and AcrB favor the viability of the intracellular bacteria. Finally, we were able to further confirm the requirement of the AcrB transporter activity for intraepithelial survival by using a specific EP inhibitor. Overall, the data from the present study expand the role of the AcrAB pump to an important human pathogen, such as Shigella, and add insights into the mechanism governing the Shigella infection process.

An automated microscopy workflow to study Shigella-neutrophil interactions and antibiotic efficacy in vivo.

Shigella are Gram-negative bacterial pathogens responsible for bacillary dysentery (also called shigellosis). The absence of a licensed vaccine and widespread emergence of antibiotic resistance has led the World Health Organisation (WHO) to highlight Shigella as a priority pathogen requiring urgent attention. Several infection models have been useful to explore the Shigella infection process; yet, we still lack information regarding events taking place in vivo. Here, using a Shigella-zebrafish infection model and high-content microscopy, we developed an automated microscopy workflow to non-invasively study fluorescently labelled bacteria and neutrophils in vivo. We applied our workflow to antibiotic-treated zebrafish, and demonstrate that antibiotics reduce bacterial burden and not neutrophil recruitment to the hindbrain ventricle. We discovered that nalidixic acid (a bactericidal antibiotic) can work with leukocytes in an additive manner to control Shigella flexneri infection and can also restrict dissemination of Shigella sonnei from the hindbrain ventricle. We envision that our automated microscopy workflow, applied here to study the interactions between Shigella and neutrophils as well as antibiotic efficacy in zebrafish, can be useful to innovate treatments for infection control in humans.

Fatty Acids Abolish Shigella Virulence by Inhibiting Its Master Regulator, VirF.

The pathogenicity of Shigella, the intracellular pathogen responsible for human bacillary dysentery, depends on a coordinated and tightly regulated expression of its virulence determinants. This is the result of a cascade organization of its positive regulators, with VirF, a transcriptional activator belonging to the AraC-XylS family, in a pivotal position. VirF itself is submitted to several well-known regulations at the transcriptional level. In this work, we present evidence for a novel posttranslational regulatory mechanism of VirF mediated by the inhibitory interaction with specific fatty acids. By homology modeling and molecular docking analyses, we identify a jelly roll motif in the structure of ViF capable of interacting with medium-chain saturated and long-chain unsaturated fatty acids. In vitro and in vivo assays show that capric, lauric, myristoleic, palmitoleic, and sapienic acids interact effectively with the VirF protein, abolishing its transcription-promoting activity. This silences the virulence system of Shigella, leading to a drastic reduction in its ability to invade epithelial cells and proliferate in their cytoplasm. IMPORTANCE In the absence of a valid vaccine, the main therapeutic approach currently used to treat shigellosis is based on the use of antibiotics. The emergence of antibiotic resistance jeopardizes the future effectiveness of this approach. The importance of the present work resides both in the identification of a new level of posttranslational regulation of the Shigella virulence system and in the characterization of a mechanism offering new opportunities for the design of antivirulence compounds, which may change the treatment paradigm of Shigella infections by limiting the emergence of antibiotic-resistant bacteria.

Investigate the immunogenic and protective effect of trivalent chimeric protein containing IpaD-StxB-TolC antigens as a vaccine candidate against S. dysenteri and S. flexneri

BackgroundsShigella spp. causes bloody diarrhea and leads to death, especially in children. Chimeric proteins containing virulence factors can prevent Shigella infection. The purpose of this study is to investigate the immunogenic and protective effect of trivalent chimeric protein containing IpaD-StxB-TolC antigens against shiga toxin, S. dysenteri and S. flexneri in vitro and in vivo conditions. MethodsRecombinant vector was transferred to E. coli BL21. The expression of the chimeric protein was confirmed by SDS PAGE and purified using the Ni-NTA column. Mice were immunized with recombinant protein and antibody titer was evaluated by ELISA. 10, 25 and 50 LD50 of Shiga toxin neutralization was evaluated in vitro (Vero cell line) and in vivo conditions. Also, the challenge of immunized mice with 10, 25 and 50 LD50 of S. dysentery and S. flexneri was done. ResultsThe expression and purification of the recombinant protein with 60.6 kDa was done. ELISA showed increased antibody titer against the chimeric protein. MTT assay indicated that 1/8000 dilution of the sera had a 51% of cell viability against the toxin in Vero cell line. The challenge of mice immunized with toxin showed that the mice had complete protection against 10 and 25 LD50 of toxin and had 40% survival against 50 LD50. Mice receiving 10 and 25 LD50 of S. dysenteri and S. flexneri had 100% protection and in 50 LD50 the survival rate was 60 and 50%, respectively. Organ burden showed that the amount of bacterial colonization in immunized mice was 1 × 104 CFU/mL, which was significantly different from the control group. ConclusionThis study showed that chimeric proteins can create favorable immunogenicity in the host as vaccine candidates.

Zebrafish null mutants of Sept6 and Sept15 are viable but more susceptible to Shigella infection.

Septins are evolutionarily conserved GTP-binding proteins known for their roles in cell division and host defence against Shigella infection. Although septin group members are viewed to function as hetero-oligomeric complexes, the role of individual septins within these complexes or in isolation is poorly understood. Decades of work using mouse models has shown that some septins (including SEPT7) are essential for animal development, while others (including SEPT6) are dispensable, suggesting that some septins may compensate for the absence of others. The zebrafish genome encodes 19 septin genes, representing the full complement of septin groups described in mice and humans. In this report, we characterise null mutants for zebrafish Sept6 (a member of the SEPT6 group) and Sept15 (a member of the SEPT7 group) and test their role in zebrafish development and host defence against Shigella infection. We show that null mutants for Sept6 and Sept15 are both viable, and that expression of other zebrafish septins are not significantly affected by their mutation. Consistent with previous reports using knockdown of Sept2, Sept7b, and Sept15, we show that Sept6 and Sept15 are required for host defence against Shigella infection. These results highlight Shigella infection of zebrafish as a powerful system to study the role of individual septins in vivo.

A22 USING INTESTINAL ORGANOIDS TO CHARACTERIZE THE NAIP-NLRC4 INFLAMMASOME RESPONSE IN THE INTESTINAL EPITHELIUM

Abstract Background The NAIP-NLRC4 inflammasome is an important innate defence mechanism in intestinal epithelial cells (IECs) that protects the gut from invasive pathogens. NAIP-NLRC4 activation triggers pyroptotic cell death, releases active interleukin (IL)-18, and promotes the expulsion of infected enterocytes. Dysregulated inflammasomes result in exaggerated inflammation of the intestinal mucosa which is characterisitic of inflammatory bowel disease (IBD). There is increasing evidence that inflammasomes in IEC behave differently than in immune cells. However, a majority of inflammasome pathway characterization is studied immune cells. In addition, NAIP-NLRC4 activity in macrophages has been shown to be important for regulating the expression of nitric oxide (Nos2). The potential transcriptomic impact of NAIP-NLRC4 activity in IECs has yet to be explored. Purpose We will use intestinal orgnaoids to systematically characterize the NAIP-NLRC4 inflammasome pathway and identify the transcriptomic impact of NAIP-NLRC4 activity in IECs. To better define the role of NAIP-NLRC4 activity during a physiological infection, we have developed a novel ex vivo model of Shigella infection in 3D organoids. Method Organoids were derived from the ileal crypts of wild type (WT) and Nlrc4-/-, Casp1-/-, Pycard-/-, or Tlr5-/- mice and stimulated with Pam3CSk4, LPS, or flagellin. Inflammasome activation was assessed by Western blot (WB) and propidium iodide uptake. WT and Nlrc4-/- organoids were infected with WT or a non-invasive Shigella mutant and the inflammasome response was evaluated by WB and a colony forming unit assay. WT and Nlrc4-/- organoids were stimulated with flagellin and gene expression was assessed by RT-qPCR. Result(s) Basolateral organoid stimulation with bacterial ligands revealed a novel response of IECs to bacterial flagellin that results in pyroptosis and IL-18 processing. Basolateral internalization of flagellin occurred in a TLR5-independent manner. Inflammasome activation by flagellin was fully abrogated in Nlrc4-/- and Casp1-/- organoids while only IL-18 processing was affected in Pycard-/- organoids. Infection with only WT Shigella induced inflammasome activation in an NLRC4-dependent manner. Interestingly, flagellin stimulation of WT but not Nlrc4-/- organoids led to increased expression of Nos2. Furthermore, Nlrc4-/- organoids had significantly lower expression levels of cytokine genes Ccl20, Cxcl1, and Tnf following inflammasome activation. Conclusion(s) Our study demonstrates an integral role for epithelial NAIP-NLRC4 inflammasomes in the response to bacterial flagellin and Shigella infection. We have uncovered a novel response of IECs to basolateral flagellin stimulation and revealed that NAIP-NLRC4 is important for the transcriptional regulation of inflammatory genes. Further work will examine how NAIP-NLRC4 activation controls inflammation and epithelial integrity by analyzing the NLRC4-dependent transcriptome and the effects on cell proliferation, differentiation, and barrier integrity. Please acknowledge all funding agencies by checking the applicable boxes below CCC, CIHR Disclosure of Interest None Declared

Sensitivity and predictive value of dysentery in diagnosing shigellosis among under five children in Zambia.

Shigella is a leading cause of bacterial diarrhea morbidity and mortality affecting mainly children under five in the developing world. In Zambia, Shigella has a high prevalence of 34.7% in children with diarrhea and an attributable fraction of 6.7% in Zambian children with moderate to severe diarrhea. Zambian diarrhea management guidelines and the health ministry reporting tool Health Management Information System (HMIS) heavily rely on the WHO clinical classification of dysentery to potentially identify and estimate the burden of Shigella in children. This reliance on clinical dysentery as a proxy to shigellosis in under five children may be resulting in gross under-estimation of shigella disease burden in Zambia. We used existing laboratory and clinical data to examine the sensitivity and predictive value of dysentery to correctly identify Shigella infection in under five children with PCR confirmed Shigella infection in Lusaka and Ndola districts, Zambia. Clinical dysentery had a sensitivity of 8.5% (34/401) in identifying under five children with Shigella by stool PCR. Dysentery was able to correctly classify Shigella in 34 of 68 bloody stool samples giving a corresponding positive predictive value of 50%. Of the 1087 with non-bloody diarrhea, 720 did not have Shigella giving a negative predictive value of 66.2%. Use of clinical dysentery as a screening symptom for Shigella infection in children under five presenting with moderate to severe diarrhea has low sensitivity and low positive predictive value respectively. Clinical dysentery as a screening symptom for Shigella contributes to gross under diagnosis and reporting of Shigella infection among under five children in Zambia. Further research is required to better inform practice on more accurate methods or tools to use in support of routine diagnosis, particularly in low middle-income settings where laboratory diagnosis remains a challenge.

Spatiotemporal variation in risk of Shigella infection in childhood: a global risk mapping and prediction model using individual participant data

Diarrhoeal disease is a leading cause of childhood illness and death globally, and Shigella is a major aetiological contributor for which a vaccine might soon be available. The primary objective of this study was to model the spatiotemporal variation in paediatric Shigella infection and map its predicted prevalence across low-income and middle-income countries (LMICs). Individual participant data for Shigella positivity in stool samples were sourced from multiple LMIC-based studies of children aged 59 months or younger. Covariates included household-level and participant-level factors ascertained by study investigators and environmental and hydrometeorological variables extracted from various data products at georeferenced child locations. Multivariate models were fitted and prevalence predictions obtained by syndrome and age stratum. 20 studies from 23 countries (including locations in Central America and South America, sub-Saharan Africa, and south and southeast Asia) contributed 66 563 sample results. Age, symptom status, and study design contributed most to model performance followed by temperature, wind speed, relative humidity, and soil moisture. Probability of Shigella infection exceeded 20% when both precipitation and soil moisture were above average and had a 43% peak in uncomplicated diarrhoea cases at 33°C temperatures, above which it decreased. Compared with unimproved sanitation, improved sanitation decreased the odds of Shigella infection by 19% (odds ratio [OR]=0·81 [95% CI 0·76-0·86]) and open defecation decreased them by 18% (OR=0·82 [0·76-0·88]). The distribution of Shigella is more sensitive to climatological factors, such as temperature, than previously recognised. Conditions in much of sub-Saharan Africa are particularly propitious for Shigella transmission, although hotspots also occur in South America and Central America, the Ganges-Brahmaputra Delta, and the island of New Guinea. These findings can inform prioritisation of populations for future vaccine trials and campaigns. NASA, National Institutes of Health-The National Institute of Allergy and Infectious Diseases, and Bill & Melinda Gates Foundation.

Facile synthesis of multi-faceted, biomimetic and cross-protective nanoparticle-based vaccines for drug-resistant Shigella: a flexible platform technology

BackgroundNo commercial vaccines are available against drug-resistant Shigella due to serotype-specific/narrow-range of protection. Nanoparticle-based biomimetic vaccines involving stable, conserved, immunogenic proteins fabricated using facile chemistries can help formulate a translatable cross-protective Shigella vaccine. Such systems can also negate cold-chain transportation/storage thus overcoming challenges prevalent in various settings.MethodsWe explored facile development of biomimetic poly (lactide-co-glycolide)/PLGA 50:50 based nanovaccines (NVs), encapsulating conserved stabilized antigen(s)/immunostimulant of S. dysenteriae 1 origin surface-modified using simple chemistries. All encapsulants (IpaC/IpaB/LPS) and nanoparticles (NPs)—bare and modified (NV), were thoroughly characterized. Effect of IpaC on cellular uptake of NPs was assessed in-vitro. Immunogenicity of the NVs was assessed in-vivo in BALB/c mice by intranasal immunization. Cross-protective efficacy was assessed by intraperitoneally challenging the immunized groups with a high dose of heterologous S. flexneri 2a and observing for visible diarrhea, weight loss and survival. Passive-protective ability of the simplest NV was assessed in the 5-day old progeny of vaccinated mice.ResultsAll the antigens and immunostimulant to be encapsulated were successfully purified and found to be stable both before and after encapsulation into NPs. The ~ 300 nm sized NPs with a zeta potential of ~ − 25 mV released ~ 60% antigen by 14th day suggesting an appropriate delivery kinetics. The NPs could be successfully surface-modified with IpaC and/or CpG DNA. In vitro experiments revealed that the presence of IpaC can significantly increase cellular uptake of NPs. All NVs were found to be cytocompatible and highly immunogenic. Antibodies in sera of NV-immunized mice could recognize heterologous Shigella. Immunized sera also showed high antibody and cytokine response. The immunized groups were protected from diarrhea and weight loss with ~ 70–80% survival upon heterologous Shigella challenge. The simplest NV showed ~ 88% survival in neonates.ConclusionsFacile formulation of biomimetic NVs can result in significant cross-protection. Further, passive protection in neonates suggest that parental immunization could protect infants, the most vulnerable group in context of Shigella infection. Non-invasive route of vaccination can also lead to greater patient compliance making it amenable for mass-immunization. Overall, our work contributes towards a yet to be reported platform technology for facile development of cross-protective Shigella vaccines.Graphical

A hierarchy of cell death pathways confers layered resistance to shigellosis in mice.

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease driven by bacterial colonization of colonic intestinal epithelial cells. Vertebrates have evolved programmed cell death pathways that sense invasive enteric pathogens and eliminate their intracellular niche. Previously we reported that genetic removal of one such pathway, the NAIP-NLRC4 inflammasome, is sufficient to convert mice from resistant to susceptible to oral Shigella flexneri challenge (Mitchell et al., 2020). Here, we investigate the protective role of additional cell death pathways during oral mouse Shigella infection. We find that the Caspase-11 inflammasome, which senses Shigella LPS, restricts Shigella colonization of the intestinal epithelium in the absence of NAIP-NLRC4. However, this protection is limited when Shigella expresses OspC3, an effector that antagonizes Caspase-11 activity. TNFα, a cytokine that activates Caspase-8-dependent apoptosis, also provides potent protection from Shigella colonization of the intestinal epithelium when mice lack both NAIP-NLRC4 and Caspase-11. The combined genetic removal of Caspases-1, -11, and -8 renders mice hyper-susceptible to oral Shigella infection. Our findings uncover a layered hierarchy of cell death pathways that limit the ability of an invasive gastrointestinal pathogen to cause disease.

Identification and antibiotic pattern analysis of bacillary dysentery causing bacteria isolated from stool samples of infected patients.

The online version contains supplementary material available at 10.1007/s11756-022-01299-x.

2005. Direct Hospitalization Costs Associated with Resistant and Susceptible Salmonella and Shigella Infections in the United States — 2012–2019

Abstract Background Foodborne illnesses cost ∼$90 billion in the United States annually. Antibiotic-resistant infections may be more difficult to treat and can be associated with severe outcomes, leading to higher costs and longer length of stay (LOS). We described LOS and costs for hospital admissions for Salmonella and Shigella infections by resistance status. Methods We reviewed all inpatient admissions from ∼300 hospitals in the Premier Healthcare database during 2012–2019 and included records with culture-confirmed nontyphoidal Salmonella (NTS), typhoidal Salmonella (serotypes Typhi and Paratyphi A, B, and C), or Shigella. We classified infections as resistant if laboratory testing found resistance to ≥1 drug in the clinically important antibiotic categories (fluoroquinolones, macrolides, penicillins, folate pathway antagonists, or third-generation cephalosporins) or susceptible otherwise. We used the Wilcoxon rank sum test to assess difference in median LOS and cost of patient hospitalization for resistant vs. susceptible infections by pathogen type. Results Of 4,168 persons hospitalized, about half (n=2,129, 51%) were female; median age was 52 [IQR: 26‒68] years; 29% (n=1,217) of infections were resistant. For NTS infections, median LOS was 4 [IQR: 3‒7] days for resistant and 4 [IQR: 3‒6] (p=0.04) for susceptible; for typhoidal Salmonella, 6 [IQR: 5‒8] for resistant and 5 [IQR: 3‒8] for susceptible (p=0.15); and for Shigella, 3 [IQR: 2‒4] for both (p=0.09). Median costs were highest for typhoidal Salmonella ($12,577 resistant vs. $9,238 susceptible; p=0.21), followed by NTS ($7,179 vs $6,421, p=0.01) and Shigella ($4,975 vs $5,335; p=0.26). Conclusion Hospitalization costs were higher for patients with some resistant infections. Longer LOS may contribute to higher costs; however, it is not clear whether other pathogen or patient factors (e.g., comorbidities) also contributed. Additional data and adjusted analyses may provide insight into reasons for the cost difference. If resistant infections are shown to be the cause, our findings support that public health interventions to limit the spread of resistant infections could reduce costs. Disclosures All Authors: No reported disclosures.

PHYTOCHEMICAL AND PHARMACOLOGICAL STUDY ON SELECTED INDONESIAN WEEDS EXTRACTS: A NOVEL INSIGHT TO ANTI-SHIGELLOSIS

Objective: Elephant grass (Pennisetum purpureum S.), weed grass (Imperata cylindrica L.), pearl grass (Hedyotis corymbosa L.) and nut grass (Cyperus rotundus L.) are selected weeds found in Indonesia which have been used as ruminants feeding with a complete diet component and evidently reported that bioactive contents of weeds provide more protection to microbial attack than that of crops. This has led to an increase interest in the investigation of weed extracts as anti-shigellosis agents for humans and animals, but there is still no data regarding on phytochemical and pharmacological of our selected weeds as an anti-shigellosis. Therefore, the objectives of this study was to analyze phytochemical and anti-shigellosis properties of those selected weeds towards sensitive (S) and resistant S. dysentriae (R) strains of ampicillin, chloramphenicol, and cotrimoxazoles. Methods: Phytochemical screening was done using the standard method and further analyzed by thin-layer chromatography (TLC). The anti-shigellosis activity was evaluated using the agar diffusion method; meanwhile, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) value was determined using the microdilution method. Results: In general, weeds contain flavonoids, steroid, and quinone compounds. The resulted anti-shigellosis showed that all weed extracts produced higher inhibition to sensitive than resistant strains. The MIC-MBC values of each weed on sensitive and resistant, respectively, were as follow: P. purpureum S (S=≥1.25%; R=≥2.5% w/v); I. cylindrica (S=≥5.0%; R=≥ 2.5-10.0%w/v); H. corymbosa (S=≥2.5%; R=≥2.5-10%w/v); and C. rotundus (S=≥2.5-5.0%; R=≥5.0-10%w/v). From these data, all of these weeds have the potential to complement antibiotics that are no longer effective in the treatment of shigella infections. Conclusion: In summary, P. purpureum extract could be promoted as a novel supplement phytopharmaceutical for the treatment of bacillary dysentery.

Prevalence of Multidrug-Resistant and Extended-Spectrum Beta-Lactamase-Producing Shigella Species in Asia: A Systematic Review and Meta-Analysis.

Shigellosis remains one of the leading causes of morbidity and mortality worldwide and is the second leading cause of diarrheal mortality among all age groups. However, the global emergence of antimicrobial-resistant Shigella strains, limiting the choice of effective drugs for shigellosis, has become the major challenge in the treatment of Shigella infections. The aim of this systematic review and meta-analysis was to provide an updated picture of the prevalence of antimicrobial-resistant Shigella species in Asia. A comprehensive and systematic search was performed on three electronic databases (PubMed, ScienceDirect and Scopus), in which 63 eligible studies published between 2010 and 2022 were identified. From our meta-analysis of proportions using a random-effects model, the overall prevalence of Shigella spp. in Asian patients was estimated to be 8.0% (95% CI: 5.5-10.5). The pooled prevalence rates of multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL)-producing Shigella strains were 68.7% (95% CI: 59.9-77.5) and 23.9% (95% CI: 12.9-34.8), respectively. Concerning recommended antimicrobial drugs for Shigella, the prevalence of resistance was highest for ciprofloxacin (29.8%) and azithromycin (29.2%), followed by ceftriaxone (23.8%), in spite of their importance as first- and second-line treatments for shigellosis. In contrast, resistance to carbapenems, such as ertapenem (0.0%), imipenem (0.1%) and meropenem (0.0%), was almost non-existent among the 49 tested antibiotics. The significantly high prevalence estimation suggests that the multidrug-resistant Shigella is a pressing threat to public health worthy of careful and justified interventions. Effective antibiotic treatment strategies, which may lead to better outcomes for the control and treatment of shigellosis in Asia, are essential.

Selective autophagy of RIPosomes maintains innate immune homeostasis during bacterial infection.

The NOD1/2-RIPK2 is a key cytosolic signaling complex that activates NF-κB pro-inflammatory response against invading pathogens. However, uncontrolled NF-κB signaling can cause tissue damage leading to chronic diseases. The mechanisms by which the NODs-RIPK2-NF-κB innate immune axis is activated and resolved remain poorly understood. Here, we demonstrate that bacterial infection induces the formation of endogenous RIPK2 oligomers (RIPosomes) that are self-assembling entities that coat the bacteria to induce NF-κB response. Next, we show that autophagy proteins IRGM and p62/SQSTM1 physically interact with NOD1/2, RIPK2 and RIPosomes to promote their selective autophagy and limit NF-κB activation. IRGM suppresses RIPK2-dependent pro-inflammatory programs induced by Shigella and Salmonella. Consistently, the therapeutic inhibition of RIPK2 ameliorates Shigella infection- and DSS-induced gut inflammation in Irgm1 KO mice. This study identifies a unique mechanism where the innate immune proteins and autophagy machinery are recruited together to the bacteria for defense as well as for maintaining immune homeostasis.

Promising directions for vaccine development to prevent shigellosis

Shigellosis (bacterial dysentery) is an acute infectious disease caused by Shigella spp., members of the Enterobacteriaceae family. The disease has the highest mortality rate amongst bacterial enteric infections. A considerable proportion of Shigella infections occur in children under the age of five. In 2017, WHO included Shigella spp. strains into the list of “priority pathogens” that are resistant to most antibiotics and pose a threat to global public health. This provided a stimulus for the development of new antibiotics to treat shigellosis. Apart from the creation of new antimicrobial therapies for Shigella infections, an important role in fighting against shigellosis belongs to the preventative measures set out in WHO’s Immunisation Agenda 2030. These include sanitation, hygiene, consumption of clean water, and vaccination. The development of Shigella vaccines has been a priority of the WHO programme for more than 20 years. The aim of the study was to analyse promising approaches to Shigella vaccine development. According to the analysis of literature, only one vaccine against shigellosis has been approved so far—Shigellvac, the Russian polysaccharide dysentery vaccine against Shigella sonnei. This study covers a number of vaccine candidates (whole-cell, polysaccharide, polysaccharide conjugate, protein antigen-based vaccines, etc.) that are at different stages of clinical trials. The importance of researching combination (multivalent) vaccines against Shigellа spp. and other enteric pathogens is noted. However, the authors consider subunit vaccines based on Ipa proteins, providing broad cross-protection against Shigellа spp., and conjugate polyvalent vaccines for children under 5 the most promising for further development.