BECC-engineered live-attenuated Shigella vaccine candidates display reduced endotoxicity with robust immunogenicity in mice.

Shigella spp. infection contributes significantly to the global disease burden, primarily affecting young children in developing countries. Currently, there are no FDA-approved vaccines against Shigella, and the prevalence of antibiotic resistance is increasing, making therapeutic options limited. Live-attenuated vaccine strains WRSs2 (S. sonnei) and WRSf2G12 (S. flexneri 2a) are highly immunogenic, making them promising vaccine candidates, but possess an inflammatory lipid A structure on their lipopolysaccharide (LPS; also known as endotoxin). Here, we utilized bacterial enzymatic combinatorial chemistry (BECC) to ectopically express lipid A modifying enzymes in WRSs2 and WRSf2G12, as well as their respective wild-type strains, generating targeted lipid A modifications across the Shigella backgrounds. Dephosphorylation of lipid A, rather than deacylation, reduced LPS-induced TLR4 signaling in vitro and dampened endotoxic effects in vivo. These BECC-modified vaccine strains retained the phenotypic traits of their parental strains, such as invasion of epithelial cells and immunogenicity in mice without adverse endotoxicity. Overall, our observations suggest that BECC-engineered live attenuated vaccines are a promising approach to safe and effective Shigella vaccines.

Antibiotic resistance of enteropathogenic bacteria in a teaching hospital in North Khuzestan during a three-year period.

Introduction: Gastrointestinal infections affect many people annually. The most common bacterial agents involved in these infections are enteropathogenic bacteria and in the continuation of using broad-spectrum antibiotics, Clostridium difficile-associated diarrhea is involved, especially in hospitalized patients. The aim of the present study was to investigate the pattern of antibiotic resistance among enteropathogenic bacteria. Materials and Methods: In this cross-sectional study, 163 samples of patients with diarrhea in Dezful Ganjavian Hospital were examined. The samples were cultured in MacConkey, Hektoen enteric agar and GN broth, and cycloserine cefoxitin fructose agar media and incubated under standard conditions. In order to identify enteropathogenic bacteria, biochemical tests and serological confirmatory tests were used. Antibiotic resistance pattern of the isolates was investigated by Kirby-Bauer disk diffusion susceptibility test. Results: The frequency of pathogenic bacteria includes 41.1% of Shigella flexneri, followed by 41.1% of S. sonnei, 6.7% of Enteropathogenic E. coli, 5.5% of Salmonella enterica Serogroup B, and 5.5% of Shigella dysenteriae. The results revealed a total of 46 patients with orders regarding C. difficile culture, no C. difficile was isolated from the samples. The studied isolates showed the highest resistance to trimethoprim-sulfamethoxazole, and ceftriaxone (88.3%), and the most effective antibiotic in the treatment of patients was ciprofloxacin with 86% sensitivity. Conclusion: Susceptibility to antibiotics was different among the isolates, which shows that the early identification of the infection agent and the selection of the correct antibiotic treatment are effective in improving the gastrointestinal infection and preventing the spread of the infection.

Comparative Genomics and Characterization of Shigella flexneri Isolated from Urban Wastewater.

Shigella species are a group of highly transmissible Gram-negative pathogens. Increasing reports of infection with extensively drug-resistant varieties of this stomach bug has convinced the World Health Organization to prioritize Shigella for novel therapeutic interventions. We herein coupled the whole-genome sequencing of a natural isolate of Shigella flexneri with a pangenome ana-lysis to characterize pathogen genomics within this species, which will provide us with an insight into its existing genomic diversity and highlight the root causes behind the emergence of quick vaccine escape variants. The isolated novel strain of S. flexneri contained ~4,500 protein-coding genes, 57 of which imparted resistance to antibiotics. A comparative pan-genomic ana-lysis revealed genomic variability of ~64%, the shared conservation of core genes in central metabolic processes, and the enrichment of unique/accessory genes in virulence and defense mechanisms that contributed to much of the observed antimicrobial resistance (AMR). A pathway ana-lysis of the core genome mapped 22 genes to 2 antimicrobial resistance pathways, with the bulk coding for multidrug efflux pumps and two component regulatory systems that are considered to work synergistically towards the development of resistance phenotypes. The prospective evolvability of Shigella species as witnessed by the marked difference in genomic content, the strain-specific essentiality of unique/accessory genes, and the inclusion of a potent resistance mechanism within the core genome, strengthens the possibility of novel serotypes emerging in the near future and emphasizes the importance of tracking down genomic diversity in drug/vaccine design and AMR governance.

Emergence of lineage III of Shigella sonnei ST152 belonging to a high-risk clone harboring the blaCTX-M-15 gene in Peru.

Multidrug-resistant Shigella sonnei ST152, global lineage III, is a high-risk clone, whose dissemination has limited therapeutic options for shigellosis. This study aimed to characterize two isolates of S. sonnei, which were recovered in Lima, Peru, during November 2019, exhibiting resistance to extended-spectrum cephalosporins and quinolones, and concurrently harboring blaCTX-M-15 and qnrS1 genes, in addition to mutations in gyrA-S83L. These isolates were resistant to ceftriaxone, ciprofloxacin and trimethoprim/sulfamethoxazole. The molecular analysis showed that both isolates belonged to lineage III, sublineages IIIa and IIIb. The blaCTX-M-15 gene was located in the same genetic platform as qnrS1, flanked upstream by ISKpn19, on a conjugative plasmid belonging to the IncI-γ group. To the best of our knowledge, this would be the first report on S. sonnei isolates carrying the blaCTX-M-15 gene in Peru. The global dissemination of S. sonnei ST152, co-resistant to ß-lactams and quinolones, could lead to a worrisome scenario in the event of potential acquisition of genetic resistance mechanisms to azithromycin.

Metabolic Analysis of Intracellular Pathogenic Bacteria Using NMR.

Pathogen proliferation and virulence depend on available nutrients, and these vary when the pathogen moves from outside of the host cell (extracellular) to the inside of the host cell (intracellular). Nuclear Magnetic Resonance (NMR) is a versatile analytical method, which lends itself for metabolic studies. In this chapter, we describe how 1H NMR can be combined with a cellular infection model to study the metabolic crosstalk between a bacterial pathogen and its host both in the extracellular and intracellular compartments. Central carbon metabolism is highlighted by using glucose labeled with the stable isotope 13C.

Outbreak of waterborne acute diarrheal disease in a South District village of Tripura: A public health emergency in the Northeast region of India.

Food and waterborne outbreaks are a neglected public health problem in India. However, it is important to identify the source of infection and the causative pathogen to curb the outbreak quickly and minimize mortality and morbidity. A retrospective descriptive study was conducted with a line list of 130 diarrheal cases. Epidemiological investigation and laboratory investigation were done. Data were collected from hospital case report forms as well as interviewed affected cases. A case of acute diarrheal disease was reported among the people in the village with abdominal pain, vomiting, and diarrhea from December 31, 2022 to January 3, 2023. Out of a total of 130 recorded cases, 33 stool samples were collected and were positive for Enteroaggregative Escherichia coli, Shigella flexneri 3a, and Shigella sonnei by cultural and molecular tests. The presumptive fecal pollution indicator assay indicated high coliform counts in the water samples (most probable number [MPN]-05) and the presence of Escherichia coli. The identified pathogens showed susceptibility to gentamicin and meropenem. People who used public drinking water were found to be infected with acute diarrheal disease (ADD). Quick identification of the causative pathogens and their antimicrobial resistance pattern helped correct antibiotic prescriptions and quick recovery of the patients without any deaths. Thus, a timely implementation of food and waterborne outbreak investigation is crucial to saving lives and preventing the spread of infection.

Designing a multi-epitope vaccine against Shigella dysenteriae using immuno-informatics approach.

Shigella dysenteriae has been recognized as the second most prevalent pathogen associated with diarrhea that contains blood, contributing to 12.9% of reported cases, and it is additionally responsible for approximately 200,000 deaths each year. Currently, there is no S. dysenteriae licensed vaccine. Multidrug resistance in all Shigella spp. is a growing concern. Current vaccines, such as O-polysaccharide (OPS) conjugates, are in clinical trials but are ineffective in children but protective in adults. Thus, innovative treatments and vaccines are needed to combat antibiotic resistance. In this study, we used immuno-informatics to design a new multiepitope vaccine and identified S. dysenteriae strain SD197's membrane protein targets using in-silico methods. The target protein was prioritized using membrane protein topology analysis to find membrane proteins. B and T-cell epitopes were predicted for vaccine formulation. The epitopes were shortlisted based on an IC50 value <50, antigenicity, allergenicity, and a toxicity analysis. In the final vaccine construct, a total of 8 B-cell epitopes, 12 MHC Class I epitopes, and 7 MHC Class II epitopes were identified for the Lipopolysaccharide export system permease protein LptF. Additionally, 17 MHC Class I epitopes and 14 MHC Class II epitopes were predicted for the Lipoprotein-releasing ABC transporter permease subunit LolE. These epitopes were selected and linked via KK, AAY, and GGGS linkers, respectively. To enhance the immunogenic response, RGD (arginine-glycine-aspartate) adjuvant was incorporated into the final vaccine construct. The refined vaccine structure exhibits a Ramachandran score of 91.5% and demonstrates stable interaction with TLR4. Normal Mode Analysis (NMA) reveals low eigenvalues (3.925996e-07), indicating steady and flexible molecular mobility of docked complexes. Codon optimization was carried out in an effective microbial expression system of the Escherichia coli K12 strain using the recombinant plasmid pET-28a (+). Finally, the entire in-silico analysis suggests that the suggested vaccine may induce a significant immune response against S. dysenteriae, making it a promising option for additional experimental trials.

Identification and analysis of immunoreactive proteins of Shigella flexneri in human sera and stool specimens.

Background: The method currently available to diagnose shigellosis is insensitive and has many limitations. Thus, this study was designed to identify specific antigenic protein(s) among the cell surface associated proteins (SAPs) of Shigella that would be valuable in the development of an alternative diagnostic assay for shigellosis, particularly one that could be run using a stool sample rather than serum. Methods: The SAPs of clinical isolates of S. dysenteriae, S. boydii, Shigella flexneri, and S. sonnei were extracted from an overnight culture grown at 37 °C using acidified-glycine extraction methods. Protein profiles were observed by SDS-PAGE. To determine if antibodies specific to certain Shigella SAPs were present in both sera and stool suspensions, Western blot analysis was used to detect the presence of IgA, IgG, and IgM. Results: Immunoblot analysis revealed that sera from patients infected with S. flexneri recognized 31 proteins. These SAP antigens are recognized by the host humoral response during Shigella infection. Specific antibodies against these antigens were also observed in intestinal secretions of shigellosis patients. Of these 31 S. flexneri proteins, the 35 kDa protein specifically reacted against IgA present in patients' stool suspensions. Further study illustrated the immunoreactivity of this protein in S. dysenteriae, S. boydii, and S. sonnei. This is the first report that demonstrates the presence of immunoreactive Shigella SAPs in stool suspensions. The SAPSs could be very useful in developing a simple and rapid serodiagnostic assay for shigellosis directly from stool specimens.

Cinnamaldehyde inhibits the NLRP3 inflammasome by preserving mitochondrial integrity and augmenting autophagy in Shigella sonnei-infected macrophages.

BACKGROUND: Worldwide, more than 125 million people are infected with Shigella each year and develop shigellosis. In our previous study, we provided evidence that Shigella sonnei infection triggers activation of the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome in macrophages. NLRP3 inflammasome is responsible for regulating the release of the proinflammatory cytokines interleukin (IL)-1ß and IL-18 through the protease caspase-1. Researchers and biotech companies have shown great interest in developing inhibitors of the NLRP3 inflammasome, recognizing it as a promising therapeutic target for several diseases. The leaves of Cinnamomum osmophloeum kaneh, an indigenous tree species in Taiwan, are rich in cinnamaldehyde (CA), a compound present in significant amounts. Our aim is to investigate how CA affects the activation of the NLRP3 inflammasome in S. sonnei-infected macrophages. METHODS: Macrophages were infected with S. sonnei, with or without CA. ELISA and Western blotting were employed to detect protein expression or phosphorylation levels. Flow cytometry was utilized to assess H2O2 production and mitochondrial damage. Fluorescent microscopy was used to detect cathepsin B activity and mitochondrial ROS production. Additionally, colony-forming units were employed to measure macrophage phagocytosis and bactericidal activity. RESULTS: CA inhibited the NLRP3 inflammasome in S. sonnei-infected macrophages by suppressing caspase-1 activation and reducing IL-1ß and IL-18 expression. CA also inhibited pyroptosis by decreasing caspase-11 and Gasdermin D activation. Mechanistically, CA reduced lysosomal damage and enhanced autophagy, while leaving mitochondrial damage, mitogen-activated protein kinase phosphorylation, and NF-κB activation unaffected. Furthermore, CA significantly boosted phagocytosis and the bactericidal activity of macrophages against S. sonnei, while reducing secretion of IL-6 and tumour necrosis factor following infection. CONCLUSION: CA shows promise as a nutraceutical for mitigating S. sonnei infection by diminishing inflammation and enhancing phagocytosis and the bactericidal activity of macrophages against S. sonnei.

Safety and immunogenicity of a 4-component GMMA-based Shigella vaccine in healthy European adults: Stage 1 of a randomized, controlled phase I/II clinical trial.

BACKGROUND: We report data from Stage 1 of an ongoing two-staged, phase I/II randomized clinical trial (NCT05073003) with a 4-component Generalized Modules for Membrane Antigens-based vaccine against Shigella sonnei and S. flexneri 1b, 2a and 3a (altSonflex1-2-3, GSK). METHODS: 18-50-year-old Europeans (N=102) were randomized (2:1) to receive two injections of altSonflex1-2-3 or placebo at 3- or 6-month interval. Safety and immunogenicity were assessed at pre-specified timepoints. RESULTS: The most common solicited administration-site event (until 7 days post-each injection) and unsolicited adverse event (until 28 days post-each injection) were pain (altSonflex1-2-3: 97.1%; Placebo: 58.8%) and headache (32.4%; 23.5%), respectively. All serotype-specific functional IgG antibodies peaked 14-28 days post-injection 1 and remained substantially higher than pre-vaccination at 3 or 6 months post-vaccination; the second injection did not boost but restored the initial immune response. The highest seroresponse rates (≥4-fold increase in titers over baseline) were obtained against S. flexneri 2a (ELISA: post-injection 1: 91.0%; post-injection 2 [Day {D}113; D197]: 100%; 97.0%; serum bactericidal activity (SBA): post-injection 1: 94.4%; post-injection 2: 85.7%; 88.9%) followed by S. sonnei (ELISA: post-injection 1: 77.6%; post-injection 2: 84.6%; 78.8%; SBA: post-injection 1: 83.3%; post-injection 2: 71.4%; 88.9%). Immune responses against S. flexneri 1b and S. flexneri 3a, as measured by both ELISA and SBA, were numerically lower compared to those against S. sonnei and S. flexneri 2a. CONCLUSIONS: No safety signals or concerns were identified. altSonflex1-2-3 induced functional serotype-specific immune responses, allowing further clinical development in the target population.

What is the context? Shigella bacteria cause severe and often bloody diarrhea, called shigellosis, that affects mostly young children and can be life-threatening. Shigellosis is particularly common in low- and middle-income countries due to inadequate sanitation and limited access to healthcare. Since the immune response to Shigella is serotype-specific, an ideal vaccine should include multiple Shigella serotypes to ensure broad protection. What is new? We developed a novel vaccine against Shigella that includes Shigella sonnei and three prevalent Shigella flexneri serotypes. In Stage 1 (phase I) of the study, healthy European adults received two vaccine injections given 3 or 6 months apart. We found that: The vaccine was well tolerated, and no safety signals or concerns were identified.Regardless of the interval between injections, specific antibodies were elicited against all four Shigella serotypes, with highest levels against Shigella flexneri 2a and Shigella sonnei.Functional antibody levels peaked after the first injection, remaining higher than the baseline up to 6 months. A second injection did not boost responses but restored functional antibody levels to those after the first injection. What is the impact? The vaccine can now be tested in Stage 2 (phase II) of the study in Africa, a region highly affected by shigellosis.

Effect of cooking methods on nutritional value and microbial safety of edible rhinoceros beetle grubs (Oryctes sp.).

Although edible rhinoceros beetle (Oryctes sp.) larvae are popularly consumed in many countries worldwide, they are prepared using different methods such as boiling, roasting, toasting, and deep-frying, whose effect on nutritional value and microbial safety is scarcely known. Here we investigated the effect of these methods on the nutritional value and microbial safety of Oryctes sp. larvae. Our hypothesis was that cooking the grubs using the four methods had no effect on their nutritional content and microbial loads and diversity. The grubs were analyzed for proximate composition, and fatty and amino acid profiles using standard chemical procedures; and microbial safety using standard culturing procedures. Deep-frying reduced protein and carbohydrate content, but elevated fat content. Boiling lowered ash content, but increased fibre and carbohydrate composition. Roasting and toasting increased protein and ash contents, respectively. Forty fatty acids were detected in the larvae, of which levels of only five were not significantly affected by cooking method, while the levels of the others were differentially affected by the different cooking methods. Amino acid profiles and levels were largely comparable across treatments, but lysine and arginine were higher in all cooked grubs than raw form. All the cooking methods eliminated Enterobacteriaceae, Shigella sp. and Campylobacter sp. from the grubs. Except boiling, all methods reduced total viable count to safe levels. Salmonella sp. were only eliminated by toasting and roasting; while boiling promoted growth of yeast and moulds. Staphylococcus aureus levels exceeded safety limits in all the cooking methods. These findings offer guidance on the type of method to use in preparing the grubs for desired nutritional and safety outcomes.

Detecting genetic gain and loss events in terms of protein domain: Method and implementation.

Continuous gain and loss of genes are the primary driving forces of bacterial evolution and environmental adaptation. Studying bacterial evolution in terms of protein domain, which is the fundamental function and evolutionary unit of proteins, can provide a more comprehensive understanding of bacterial differentiation and phenotypic adaptation processes. Therefore, we proposed a phylogenetic tree-based method for detecting genetic gain and loss events in terms of protein domains. Specifically, the method focuses on a single domain to trace its evolution process or on multiple domains to investigate their co-evolution principles. This novel method was validated using 122 Shigella isolates. We found that the loss of a significant number of domains was likely the main driving force behind the evolution of Shigella, which could reduce energy expenditure and preserve only the most essential functions. Additionally, we observed that simultaneously gained and lost domains were often functionally related, which can facilitate and accelerate phenotypic evolutionary adaptation to the environment. All results obtained using our method agree with those of previous studies, which validates our proposed method.

Identification of therapeutic drug target of Shigella Flexneri serotype X through subtractive genomic approach and in-silico screening based on drug repurposing.

Shigellosis, induced by Shigella flexneri, constitutes a significant health burden in developing nations, particularly impacting socioeconomically disadvantaged communities. Designated as the second most prevalent cause of diarrheal illness by the World Health Organization (WHO), it precipitates an estimated 212,000 fatalities annually. Within the spectrum of S. flexneri strains, serotype X is notably pervasive and resilient, yet its comprehensive characterization remains deficient. The present investigation endeavors to discern potential pharmacological targets and repurpose existing drug compounds against S. flexneri serotype X. Employing the framework of subtractive genomics, the study interrogates the reference genome of S. flexneri Serotype X (strain 2,002,017; UP000001884) to delineate its proteome into categories of non-homologous, non-paralogous, essential, virulent, and resistant constituents, thereby facilitating the identification of therapeutic targets. Subsequently, a screening of approximately 9000 compounds from the FDA library against the identified drug target aims to delineate efficacious agents for combating S. flexneri serotype X infections. The application of subtractive genomics methodology yields prognostic insights, unveiling non-paralogous proteins (n = 4122), non-homologues (n = 1803), essential (n = 1246), drug-like (n = 389), resistant (n = 167), alongside 42 virulent proteins within the reference proteome. This iterative process culminates in the identification of Serine O-acetyltransferase as a viable drug target. Subsequent virtual screening endeavors to unearth FDA-approved medicinal compounds capable of inhibiting Serine O-acetyltransferase. Noteworthy candidates such as DB12983, DB15085, DB16098, DB16185, and DB16262 emerge, exhibiting potential for mitigating S. flexneri Serotype X. Despite the auspicious findings, diligent scrutiny is imperative to ascertain the efficacy and safety profile of the proposed drug candidates vis-à-vis S. flexneri.

Bile acid-gut microbiota imbalance in cholestasis and its long-term effect in mice.

Cholestasis is a common morbid state that may occur in different phases; however, a comprehensive evaluation of the long-term effect post-recovery is still lacking. In the hepatic cholestasis mouse model, which was induced by a temporary complete blockage of the bile duct, the stasis of bile acids and liver damage typically recovered within a short period. However, we found that the temporary hepatic cholestasis had a long-term effect on gut microbiota dysbiosis, including overgrowth of small intestinal bacteria, decreased diversity of the gut microbiota, and an overall imbalance in its composition accompanied by an elevated inflammation level. Additionally, we observed an increase in Escherichia-Shigella (represented by ASV136078), rich in virulence factors, in both small and large intestines following cholestasis. To confirm the causal role of dysregulated gut microbiota in promoting hepatic inflammation and injury, we conducted gut microbiota transplantation into germ-free mice. We found that recipient mice transplanted with feces from cholestasis mice exhibited liver inflammation, damage, and accumulation of hepatic bile acids. In conclusion, our study demonstrates that cholestasis disrupts the overall load and structural composition of the gut microbiota in mice, and these adverse effects persist after recovery from cholestatic liver injury. This finding suggests the importance of monitoring the structural composition of the gut microbiota in patients with cholestasis and during their recovery. IMPORTANCE: Our pre-clinical study using a mouse model of cholestasis underscores that cholestasis not only disrupts the equilibrium and structural configuration of the gut microbiota but also emphasizes the persistence of these adverse effects even after bile stasis restoration. This suggests the need of monitoring and initiating interventions for gut microbiota structural restoration in patients with cholestasis during and after recovery. We believe that our study contributes to novel and better understanding of the intricate interplay among bile acid homeostasis, gut microbiota, and cholestasis-associated complications. Our pre-clinical findings may provide implications for the clinical management of patients with cholestasis.

Vaccination with a Protective Ipa Protein-Containing Nanoemulsion Differentially Alters the Transcriptomic Profiles of Young and Elderly Mice following Shigella Infection.

Shigella spp. are responsible for bacillary dysentery or shigellosis transmitted via the fecal-oral route, causing significant morbidity and mortality, especially among vulnerable populations. There are currently no licensed Shigella vaccines. Shigella spp. use a type III secretion system (T3SS) to invade host cells. We have shown that L-DBF, a recombinant fusion of the T3SS needle tip (IpaD) and translocator (IpaB) proteins with the LTA1 subunit of enterotoxigenic E. coli labile toxin, is broadly protective against Shigella spp. challenge in a mouse lethal pulmonary model. Here, we assessed the effect of LDBF, formulated with a unique TLR4 agonist called BECC470 in an oil-in-water emulsion (ME), on the murine immune response in a high-risk population (young and elderly) in response to Shigella challenge. Dual RNA Sequencing captured the transcriptome during Shigella infection in vaccinated and unvaccinated mice. Both age groups were protected by the L-DBF formulation, while younger vaccinated mice exhibited more adaptive immune response gene patterns. This preliminary study provides a step toward identifying the gene expression patterns and regulatory pathways responsible for a protective immune response against Shigella. Furthermore, this study provides a measure of the challenges that need to be addressed when immunizing an aging population.

Enteric Infection-Associated Reactive Arthritis: A Systematic Review and Meta-Analysis.

Background. The objective of this systematic review and meta-analysis was to estimate the proportions of individuals infected with Campylobacter, Escherichia, Salmonella, Shigella, or Yersinia who develop reactive arthritis. Methods. A systematic review was conducted, encompassing English-language articles published before January 2024, sourced from the Embase, PubMed, Scopus, and Web of Science databases. This review included observational studies that reported the occurrence of reactive arthritis (ReA) among patients with Campylobacter, Escherichia, Salmonella, Shigella, or Yersinia infections. Data extraction was carried out independently by two reviewers. Subsequently, a random-effects meta-analysis was performed, with heterogeneity assessed using the I2 value. Additionally, meta-regression was employed to investigate the potential influence of study-level variables on the observed heterogeneity. Results. A total of 87 studies were identified; 23 reported on ReA development after Campylobacter infection, 7 reported on ReA after Escherichia infection, 30 reported ReA onset after salmonellosis, 14 reported ReA after shigellosis, and 13 reported ReA after Yersinia infection. The proportion of Campylobacter patients who developed ReA was 0.03 (95% CI [0.01, 0.06], I2 = 97.62%); the proportion of Escherichia patients who developed ReA was 0.01 (95% CI [0.00, 0.06], I2 = 92.78%); the proportion of Salmonella patients was 0.04 (95% CI [0.02, 0.08], I2 = 97.67%); the proportion of Shigella patients was 0.01 (95% CI [0.01, 0.03], I2 = 90.64%); and the proportion of Yersinia patients who developed ReA was 0.05 (95% CI [0.02, 0.13], I2 = 96%). Conclusion. A significant proportion of Salmonella, Shigella, and Yersinia cases resulted in ReA. Nonetheless, it is important to interpret the findings cautiously due to the substantial heterogeneity observed between studies.

Strain-specific Recovery of S. sonnei from Artificially Contaminated Baby Carrots: Enhancing Food-safety Investigations with a Customized Shigella Detection Method Based on Genomically predicted Antibiotic Resistance Traits.

Shigella spp. are Gram-negative gastrointestinal bacterial pathogens that cause bacillary dysentery or shigellosis in humans. Isolation of Shigella from outbreak-associated foods is often problematic due to the lack of selectivity of cultural enrichment broths. To facilitate Shigella recovery from foods, we have developed strain-specific enrichment media based on the genomically-predicted antimicrobial resistance (AMR) features of an outbreak-associated Shigella sonnei strain harboring resistance genes for streptomycin (STR) and trimethoprim (TMP). To assess performance of the method, baby carrots were artificially contaminated with the S. sonnei strain at low (2.4 CFU), medium (23.5 CFU), and high levels (235 CFU) along with 10-fold higher levels of a Shigella-inhibiting Escherichia coli strain. The target S. sonnei strain was successfully recovered from artificially-contaminated baby carrots when enriched in modified Tryptone Soya Broth (mTSB) supplemented with TMP, whereas Shigella was not recovered from Shigella broth (SB) or SB supplemented with STR. Quantitative PCR analysis indicated that supplementation of the enrichment broths with TMP or STR increased the relative proportion of S. sonnei in enrichment cultures, except at the lowest inoculation level for STR. Microbiome profiling of the baby carrot enrichment cultures conducted by 16S rRNA gene sequencing indicated that both SB-STR and mTSB-TMP repressed the growth of competing Enterobacteriaceae in the enrichment cultures, relative to SB without supplementation. Overall, improved Shigella recovery was achieved with the addition of the appropriate custom selective agent during cultural enrichments demonstrating that genomically informed custom selective enrichment of Shigella could be a valuable tool for supporting future foodborne shigellosis outbreak investigations.

Overcoming Microbial Inhibition of S. Sonnei Through the Exploitation of Genomically Predicted Antibiotic Resistance Profiles for the Development of Food Enrichment Media.

Linking outbreaks of Shigella spp. to specific foods is challenging due to poor selectivity of current enrichment media. We have previously shown that enrichment media, tailored to the genomically-predicted antimicrobial resistance (AMR) of Shiga toxigenic E. coli strains, enhances their isolation from foods. This study investigates the application of this approach for Shigella isolation. The AMR gene profiles of 21,908 published S. sonnei genomes indicated a high prevalence of genes conferring resistance to streptomycin (aadA, aph(3″)-Ib, aph(6)-Id, 92.8%), sulfonamides (sul1, sul2, 74.8%), and/or trimethoprim (dfrA, 96.2%). Genomic analysis and antibiotic susceptibility testing conducted with a panel of 17 outbreak-associated S. sonnei strains confirmed the correlation of AMR gene detection with resistance phenotypes. Supplementation of Shigella Broth (SB) with up to 400 µg/mL of trimethoprim or sulfadiazine did not suppress the growth of sensitive strains, whereas 100 µg/mL of streptomycin increased the selectivity of this broth. All three antibiotics increased the selectivity of modified Tryptone Soya Broth (mTSB). Based on these results, supplemented media formulations were developed and assessed by measuring the relative growth of S. sonnei in cultures coinoculated with a strain of bacteriocin-producing E. coli that is inhibitory to Shigella growth. S. sonnei was not recovered from cocultures grown in SB or mTSB without antibiotics. In contrast, media supplemented with streptomycin at 50 and 100 µg/mL, trimethoprim at 25 and 50 µg/mL, and sulfadiazine at 100 µg/mL increased the relative proportion of S. sonnei in postenrichment cultures. The enhanced recovery of resistant S. sonnei strains achieved in this study indicates that, in cases where genomic data are available for clinical S. sonnei isolates, customization of selective enrichment media based on AMR gene detection could be a valuable tool for supporting the investigation of foodborne shigellosis outbreaks.

Determining glycosyltransferase functional order via lethality due to accumulated O-antigen intermediates, exemplified with Shigella flexneri O-antigen biosynthesis.

The O antigen (OAg) polysaccharide is one of the most diverse surface molecules of Gram-negative bacterial pathogens. The structural classification of OAg, based on serological typing and sequence analysis, is important in epidemiology and the surveillance of outbreaks of bacterial infections. Despite the diverse chemical structures of OAg repeating units (RUs), the genetic basis of RU assembly remains poorly understood and represents a major limitation in assigning gene functions in polysaccharide biosynthesis. Here, we describe a genetic approach to interrogate the functional order of glycosyltransferases (GTs). Using Shigella flexneri as a model, we established an initial glycosyltransferase (IT)-controlled system, which allows functional order allocation of the subsequent GT in a 2-fold manner as follows: (i) first, by reporting the growth defects caused by the sequestration of UndP through disruption of late GTs and (ii) second, by comparing the molecular sizes of stalled OAg intermediates when each putative GT is disrupted. Using this approach, we demonstrate that for RfbF and RfbG, the GT involved in the assembly of S. flexneri backbone OAg RU, RfbG, is responsible for both the committed step of OAg synthesis and the third transferase for the second L-Rha. We also show that RfbF functions as the last GT to complete the S. flexneri OAg RU backbone. We propose that this simple and effective genetic approach can be also extended to define the functional order of enzymatic synthesis of other diverse polysaccharides produced both by Gram-negative and Gram-positive bacteria.IMPORTANCEThe genetic basis of enzymatic assembly of structurally diverse O antigen (OAg) repeating units (RUs) in Gram-negative pathogens is poorly understood, representing a major limitation in our understanding of gene functions for the synthesis of bacterial polysaccharides. We present a simple genetic approach to confidently assign glycosyltransferase (GT) functions and the order in which they act during assembly of the OAg RU. We employed this approach to determine the functional order of GTs involved in Shigella flexneri OAg assembly. This approach can be generally applied in interrogating GT functions encoded by other bacterial polysaccharides to advance our understanding of diverse gene functions in the biosynthesis of polysaccharides, key knowledge in advancing biosynthetic polysaccharide production.

Prevalence of Salmonella spp., Shigella spp., and intestinal parasites among food handlers working in University of Gondar student's cafeteria, Northwest Ethiopia.

Background: Food-borne infections continue to be a major public health problem at the international level. The issue becomes more serious in developing countries like Ethiopia. Objective: This study aimed to examine the prevalence of Salmonella and Shigella species and intestinal parasites, as well as antimicrobial resistance patterns and associated factors among food handlers at the University of Gondar cafeteria in northwest Ethiopia. Methods: An institutional-based cross-sectional study was conducted from February to June 2021 in the University of Gondar cafeterias. Data related to the socio-demographic characteristics and hygienic practices of study participants were collected using structured questionnaires. A total of 290 stool samples were collected from food handlers. Culture and conventional biochemical tests were used to isolate the Salmonella and the Shigella species. Wet mount, Formol-ether concentration, and Kato Katz techniques were applied to identify intestinal parasites. Additionally, drug susceptibility tests were performed using the disk diffusion method. Statistical analysis was done using SPSS version 26. Results: Of 290 food handlers' stool samples analyzed, Twenty-seven 27 (9.3%) were positive for both Salmonella and Shigella species. The prevalence of Salmonella and Shigella species was 16 (5.5%) and 11 (3.8%), respectively. Most of the isolated pathogens were resistant to tetracycline 19 (70.4%), and trimethoprim/sulphamethoxazole 19 (70.4%). The overall rate of multi-drug resistant Shigella and Salmonella isolate was 59.3%. Besides, Fifty-seven 57 (19.7%) of the participants were positive for one or more intestinal parasites. The most prevalent intestinal Parasitosis was E. histolytica/dispar 22 (7.6%), followed by G. lamblia 13 (4.5%), and Ascaris lumbricoides 11 (3.8) not washing hands after using the toilet (AOR: 4.42, 95% CI: 1.57, 10.56), and consuming unpasteurized milk (AOR: 3.14, 95% CI: 1.65, 3.96), were factors significantly associated with the prevalence of Salmonella, and Shigella infection. Similarly, not washing hands after using the toilet (AOR: 2.19, 95% CI: 1.0, 1.4), and consuming unpasteurized milk (AOR: 10.4, 95% CI: 3.8, 28.8), were factors significantly associated with the prevalence of intestinal parasites infection. Conclusion: The prevalence of intestinal parasites, Salmonella, and Shigella species was high. Therefore, it is imperative to implement a public health policy that includes ongoing microbiological surveillance.