ABSTRACT
Azithromycin-resistant shigellosis is increasing globally. This retrospective analysis of Shigella flexneri serotype 2a isolates from 2016 to 2018 in Ontario found nearly half were azithromycin (47.7%, 72/151) and ciprofloxacin (50.7%, 77/152) resistant. Moreover, 34.7% (25/72) of azithromycin-resistant isolates were also ciprofloxacin-resistant. Four isolates were ceftriaxone-resistant, although all azithromycin-resistant isolates were ceftriaxone-susceptible. Overall, 83.6% (127/152) of all S. flexneri 2a isolates were recovered from males and 97.2% (70/72) of the azithromycin-resistant cases were males. Among the azithromycin-resistant cases, some (8/72) reported international travel. Phylogenetic analysis of azithromycin-resistant isolates revealed two large male-dominated clusters, and one cluster may have been due to importation of resistant strain. Comparison of plasmids isolated from the clusters in Ontario revealed the presence of incFII plasmid with high percentage of similarity to plasmids present in global outbreaks affecting mostly males including men who have sex with men (MSM). These two large azithromycin-resistant clusters are suggestive of an outbreak among MSM, though disease exposure or sexual orientation of patients was unknown. The presence of plasmid-borne azithromycin resistance in ciprofloxacin-resistant isolates is a public health concern. Antimicrobial surveillance is important for patient management, understanding the spread of novel resistance types in local communities which sometimes is introduced by travel. We found ongoing multidrug-resistant outbreaks spanning multiple years affecting males. Reduction of future outbreaks in high-risk communities like MSM requires consorted information flow between laboratory, public health, and physicians. We impart genomic and antimicrobial characteristics of multidrug S. flexneri 2a which may serve as reference by clinicians and public health.IMPORTANCEOral ciprofloxacin and azithromycin are generally considered as the first-line therapy of shigellosis. Here, we report the emergence and transmission of azithromycin and ciprofloxacin-resistant S. flexneri serotype 2a among male adults in Ontario during 2016-2018. The percentage of azithromycin and ciprofloxacin resistance among S. flexneri 2a is higher compared to previous reports from Canada and United States. Here, we show the genetic basis of the antimicrobial resistance among these unique groups of S. flexneri 2a isolates. We describe a domestically acquired azithromycin-resistant and ciprofloxacin-resistant S. flexneri 2a lineage in Ontario. Combining whole-genome sequencing (WGS) data with travel-associated data helped in understanding dissemination and transmission. We employed WGS, which not only helped us in understanding the genetic-relationship between isolates but also mine information regarding plasmids. In the future, linking WGS, travel-related data, and clinical data can provide enhanced contact tracing and improve public-health management.
ABSTRACT
Shigella flexneri (S. flexneri) is a facultatively anaerobic gram-negative bacterium that is a member of Enterobacteriaceae. The bacterium has been known to cause mild symptoms, such as diarrhea, to more severe diseases such as hemorrhagic colitis. Fortunately, such instances of severe diseases are rare. Nevertheless, even though S. flexneri is a more benign bacterium of the Shigella genus when compared to Shigella dysenteriae, this doesn't mean that it should be neglected. In fact, the ability of this microorganism to cause inflammation of the colon or colitis and disrupt tissue architecture can allow other organisms that would otherwise be benign to cause severe complications, hence allowing said organisms to be opportunistic. Here, we would like to present a case of S. flexneri colitis resulting in bacillus bacteremia and eventually causing an inappropriate physiological host response leading to hypotension, systematic organ failure, etc., also known as septic shock. The pathogenesis and treatment of this patient will also be discussed.
ABSTRACT
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.
Subject(s)
Anti-Bacterial Agents , Biofilms , Eugenol , Shigella flexneri , Biofilms/drug effects , Biofilms/growth & development , Shigella flexneri/drug effects , Shigella flexneri/genetics , Shigella flexneri/growth & development , Shigella flexneri/physiology , Eugenol/pharmacology , Anti-Bacterial Agents/pharmacology , Quorum Sensing/drug effects , Microbial Sensitivity Tests , Dysentery, Bacillary/drug therapy , Dysentery, Bacillary/microbiology , Terpenes/pharmacologyABSTRACT
Target of Myb1 (TOM1) facilitates the transport of endosomal ubiquitinated proteins destined for lysosomal degradation; however, the mechanisms regulating TOM1 during this process remain unknown. Here, we identified an adjacent DXXLL motif-containing region to the TOM1 VHS domain, which enhances its affinity for ubiquitin and can be modulated by phosphorylation. TOM1 is an endosomal phosphatidylinositol 5-phosphate (PtdIns5P) effector under Shigella flexneri infection. We pinpointed a consensus PtdIns5P-binding motif in the VHS domain. We show that PtdIns5P binding by TOM1 is pH-dependent, similarly observed in its binding partner TOLLIP. Under acidic conditions, TOM1 retained its complex formation with TOLLIP, but was unable to bind ubiquitin. S. flexneri infection inhibits pH-dependent endosomal maturation, leading to reduced protein degradation. We propose a model wherein pumping of H+ to the cytosolic side of endosomes contributes to the accumulation of TOM1, and possibly TOLLIP, at these sites, thereby promoting PtdIns5P- and pH-dependent signaling, facilitating bacterial survival.
ABSTRACT
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.
Subject(s)
Anti-Bacterial Agents , Genome, Bacterial , Genomics , Shigella flexneri , Wastewater , Shigella flexneri/genetics , Shigella flexneri/isolation & purification , Shigella flexneri/classification , Shigella flexneri/drug effects , Genome, Bacterial/genetics , Wastewater/microbiology , Anti-Bacterial Agents/pharmacology , Phylogeny , Whole Genome Sequencing , Drug Resistance, Multiple, Bacterial/genetics , Virulence/geneticsABSTRACT
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.
Subject(s)
Anti-Bacterial Agents , Drug Repositioning , Dysentery, Bacillary , Genomics , Serogroup , Shigella flexneri , Shigella flexneri/drug effects , Shigella flexneri/genetics , Drug Repositioning/methods , Genomics/methods , Anti-Bacterial Agents/pharmacology , Dysentery, Bacillary/drug therapy , Dysentery, Bacillary/microbiology , Humans , Genome, Bacterial , Computer Simulation , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
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.
Subject(s)
Bacterial Proteins , Glycosyltransferases , O Antigens , Shigella flexneri , Shigella flexneri/genetics , Shigella flexneri/enzymology , Shigella flexneri/metabolism , O Antigens/biosynthesis , O Antigens/genetics , O Antigens/metabolism , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
The presence of enteric pathogens in produce can serve as a significant means of transmitting infections to consumers. Notably, tomatoes, as a type of produce, have been implicated in outbreaks caused by various human pathogens, such as Salmonella enterica and pathogenic Escherichia coli. However, the survival characteristics of Shigella spp. in tomatoes have not been thoroughly investigated. In this study, we assess the survival of S. flexneri 2a in two distinct varieties of post-harvested tomatoes. S. flexneri 2a was used to inoculate both regular-sized Vine tomatoes and cherry-type Mini Plum tomatoes. Our findings reveal no significant difference in Shigella survival in the pericarp of both varieties on day 2 post-inoculation. However, a significant disparity emerges on day 6, where all recovered Shigella colonies exclusively belong to the Mini Plum variety, with none associated with the Vine type. When Shigella was inoculated into the locular cavity (deep inoculation), no significant difference between varieties was observed. Additionally, we investigate the potential role of the SRL pathogenicity island (SRL PAI) in the survival and fitness of S. flexneri 2a in post-harvested tomatoes. Our results indicate that while the SRL PAI is not linked to the survival of the strains in tomato, it does impact their fitness. These findings underscore the variability in Shigella strains' survival capabilities depending on the tomato variety, highlighting the importance of understanding Shigella ecology beyond the human host and identifying molecular determinants influencing bacterial survival to mitigate the risk of future outbreaks. The significance of this data on Shigella persistence in fresh vegetables should not be underestimated, as even a small number of Shigella cells can pose a threat to the health of individuals.
ABSTRACT
Bacterial enteritis has a substantial role in contributing to a large portion of the global disease burden and serves as a major cause of newborn mortality. Despite advancements gained from current animal and cell models in improving our understanding of pathogens, their widespread application is hindered by apparent drawbacks. Therefore, more precise models are imperatively required to develop more accurate studies on host-pathogen interactions and drug discovery. Since the emergence of intestinal organoids, massive studies utilizing organoids have been conducted to study the pathogenesis of bacterial enteritis, revealing new mechanisms and validating established ones. In this review, we focus on the advancements of several bacterial pathogenesis mechanisms observed in intestinal organoid/enteroid models, exploring the host response and bacterial effectors during the infection process. Finally, we address the features that warrant additional investigation or could be enhanced in existing organoid models in order to guide future research endeavors.
Subject(s)
Bacterial Infections , Enteritis , Animals , Intestines/microbiology , Bacteria , OrganoidsABSTRACT
Pathogen infections including Shigella flexneri have posed a significant threat to human health for numerous years. Although culturing and qPCR were the gold standards for pathogen detection, time-consuming and instrument-dependent restrict their application in rapid diagnosis and economically less-developed regions. Thus, it is urgently needed to develop rapid, simple, sensitive, accurate, and low-cost detection methods for pathogen detection. In this study, an immunomagnetic beads-recombinase polymerase amplification-CRISPR/Cas12a (IMB-RPA-CRISPR/Cas12a) method was built based on a cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of S. flexneri in the laboratory. Firstly, S. flexneri was specifically captured and enriched by IMB (Shigella antibody-coated magnetic beads), and the genomic DNA was released and used as the template in the RPA reaction. Then, the RPA products were mixed with the pre-loaded CRISPR/Cas12a for fluorescence visualization. The results were observed by naked eyes under LED blue light, with a sensitivity of 5 CFU/mL in a time of 70 min. With no specialized equipment or complicated technical requirements, the IMB-RPA-CRISPR/Cas12a diagnostic method can be used for visual, rapid, and simple detection of S. flexneri and can be easily adapted to monitoring other pathogens.
Subject(s)
Antibodies , Shigella flexneri , Humans , Blue Light , Fluorescence , RecombinasesABSTRACT
We report the detection of OXA-181 carbapenemase in an azithromycin-resistant Shigella spp. bacteria in an immunocompromised patient. The emergence of OXA-181 in Shigella spp. bacteria raises concerns about the global dissemination of carbapenem resistance in Enterobacterales and its implications for the treatment of infections caused by Shigella bacteria.
Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Dysentery, Bacillary , Microbial Sensitivity Tests , Shigella flexneri , beta-Lactamases , Shigella flexneri/drug effects , Shigella flexneri/isolation & purification , Shigella flexneri/enzymology , Shigella flexneri/genetics , Humans , beta-Lactamases/genetics , beta-Lactamases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Dysentery, Bacillary/microbiology , Dysentery, Bacillary/diagnosis , Dysentery, Bacillary/drug therapy , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Male , Immunocompromised Host , Drug Resistance, Bacterial , Azithromycin/pharmacology , Azithromycin/therapeutic useABSTRACT
Shigella flexneri causes severe diarrheal disease worldwide. While many aspects of pathogenesis have been elucidated, significant knowledge gaps remain regarding the role of putative chromosomally-encoded virulence genes. The uncharacterized sap gene encoded on the chromosome has significant nucleotide sequence identity to the fluffy (flu) antigen 43 autotransporter gene in pathogenic Escherichia coli. Here, we constructed a Δsap mutant in S. flexneri strain 2457T and examined the effects of this mutation on bacterial cell aggregation, biofilm formation, and adherence to colonic epithelial cells. Analyses included the use of growth media supplemented with glucose and bile salts to replicate small intestinal signals encountered by S. flexneri. Deletion of the sap gene in 2457T affected epithelial cell adherence, resulted in quicker bacterial cell aggregation, but did not affect biofilm formation. This work highlights a functional role for the sap gene in S. flexneri pathogenesis and further demonstrates the importance of using relevant and appropriate gastrointestinal signals to characterize virulence genes of enteropathogenic bacteria.
Subject(s)
Gastrointestinal Microbiome , Type V Secretion Systems , Type V Secretion Systems/genetics , Shigella flexneri/genetics , Epithelial Cells/microbiology , Mutation , Escherichia coli , Bacterial Proteins/geneticsABSTRACT
The objective of the current study was to examine the antimicrobial, anti-adhesion, and anti-invasion properties of various concentrations of condition media obtained from adipose mesenchymal stem cells (AD-MSCs CM) against Shigella flexneri (S. flexneri). AD-MSCs characterization and antimicrobial assay were performed using flow cytometry and microdilution by colony counting, respectively. For evaluating adhesion and invasion, Caco-2 cells were infected by S. flexneri at three different multiplicities of infection (MOIs of 1, 10, and 50) and then treated with DMEM medium and AD-MSCs CM. The inhibitory effect of AD-MSCs CM was assessed after 24 and 48 h of treatment by CFU (colony-forming unit) counting. A total of 84, 65, and 56% reduction in the adhesion rate of S. flexneri to Caco-2 cells treated with AD-MSCs CM were observed at MOIs of 1, 10, and 50, respectively. While S. flexneri at MOI:1 had no invasive effect on Caco-2 cells, convincing invasion was detected at MOIs of 10 and 50, showing a significant decrease following treatment with AD-MSCs CM. The current study results open new insights into AD-MSCs CM as a new non-antibiotic therapeutic candidate for S. flexneri infections.
Subject(s)
Anti-Infective Agents , Mesenchymal Stem Cells , Humans , Shigella flexneri , Caco-2 Cells , ObesityABSTRACT
This study announces the genome sequence of the Shigella flexneri MTR_GR_V146 strain isolated from a tomato (Solanum lycopersicum) sample in Bangladesh. This strain has a 4,624,521 bp genome length (coverage: 73.07×), 2 CRISPR arrays, 1 plasmid, 52 predicted antibiotic resistance genes, and 53 virulence factor genes.
ABSTRACT
Background: Shigellosis mainly affects children under 5 years of age living in low- and middle-income countries, who are the target population for vaccination. There are, however, limited data available to define the appropriate timing for vaccine administration in this age group. Information on antibody responses following natural infection, proxy for exposure, could help guide vaccination strategies. Methods: We undertook a retrospective analysis of antibodies to five of the most prevalent Shigella serotypes among children aged <5 years in Kenya. Serum samples from a cross-sectional serosurvey in three Kenyan sites (Nairobi, Siaya, and Kilifi) were analyzed by standardized ELISA to measure IgG against Shigella sonnei and Shigella flexneri 1b, 2a, 3a, and 6. We identified factors associated with seropositivity to each Shigella serotype, including seropositivity to other Shigella serotypes. Results: A total of 474 samples, one for each participant, were analyzed: Nairobi (n = 169), Siaya (n = 185), and Kilifi (n = 120). The median age of the participants was 13.4 months (IQR 7.0-35.6), and the male:female ratio was 1:1. Geometric mean concentrations (GMCs) for each serotype increased with age, mostly in the second year of life. The overall seroprevalence of IgG antibodies increased with age except for S. flexneri 6 which was high across all age subgroups. In the second year of life, there was a statistically significant increase of antibody GMCs against all five serotypes (p = 0.01-0.0001) and a significant increase of seroprevalence for S. flexneri 2a (p = 0.006), S. flexneri 3a (p = 0.006), and S. sonnei (p = 0.05) compared with the second part of the first year of life. Among all possible pairwise comparisons of antibody seropositivity, there was a significant association between S. flexneri 1b and 2a (OR = 6.75, 95% CI 3-14, p < 0.001) and between S. flexneri 1b and 3a (OR = 23.85, 95% CI 11-54, p < 0.001). Conclusion: Children living in low- and middle-income settings such as Kenya are exposed to Shigella infection starting from the first year of life and acquire serotype-specific antibodies against multiple serotypes. The data from this study suggest that Shigella vaccination should be targeted to infants, ideally at 6 or at least 9 months of age, to ensure children are protected in the second year of life when exposure significantly increases.
Subject(s)
Dysentery, Bacillary , Shigella , Infant , Child , Humans , Male , Female , Child, Preschool , Kenya/epidemiology , Serogroup , Immunoglobulin G , Retrospective Studies , Seroepidemiologic Studies , Cross-Sectional Studies , VaccinationABSTRACT
Under stress conditions, translationally stalled mRNA and associated proteins undergo liquid-liquid phase separation and condense into cytoplasmic foci called stress granules (SGs). Many viruses hijack SGs for their pathogenesis; however, whether pathogenic bacteria also exploit this pathway remains unknown. Here, we report that members of the OspC family of Shigella flexneri induce SG formation in infected cells. Mechanistically, the OspC effectors target multiple subunits of the host translation initiation factor 3 complex by ADP-riboxanation. The modification of eIF3 leads to translational arrest and thus the formation of SGs. Furthermore, OspC-mediated SGs are beneficial for S. flexneri replication within infected host cells, and bacterial strains unable to induce SGs are attenuated for virulence in a murine model of infection. Our findings reveal a mechanism by which bacterial pathogens induce SG assembly by inactivating host translational machinery and promote bacterial proliferation in host cells.
Subject(s)
Eukaryotic Initiation Factor-3 , Shigella , Animals , Mice , Stress Granules , Cytoplasm , Shigella flexneriABSTRACT
Shigella typically causes gastrointestinal infections, and extra-intestinal manifestations are rare. We report the first known case of pyogenic cervical spondylitis co-infected with Escherichia coli and Shigella flexneri, highlighting the diagnostic challenges and clinical implications. A 53-year-old woman presented with neck pain for one month. MRI revealed C6 and C7 vertebrae abscesses. The patient underwent anterior cervical debridement and bone-graft fusion. Intraoperative pus culture grew Escherichia coli, while metagenomic next-generation sequencing detected both Escherichia coli and Shigella species. Intravenous imipenem 500 mg every 6 h was administered, leading to full wound healing at a 6-month follow-up. This case emphasizes the importance of considering Shigella infection in the differential diagnosis of pyogenic spondylitis and demonstrates the utility of a multi-pronged diagnostic approach.
ABSTRACT
Falafel is a popular breakfast food in the Middle East that has been recently involved in several outbreaks of foodborne illnesses. The aim of the study was to explore the growth behavior of Salmonella enterica, Escherichia coli O157:H7, Shigella sonnie, Shigella flexneri, Listeria monocytogenes and Staphylococcus aureus in falafel paste (FP) under different storage temperatures (4, 10, or 24 °C) for 14 days. FP (pH = 6.2, aw = 0.96) was inoculated with 5.0 to 6.0 log CFU/g of each of the pathogens separately. Salmonella spp. significantly declined by 1.5 log at 4 °C but grew significantly by ca. 2 and 4 log at 10 and 24 °C, respectively after 14 days. E. coli O157:H7 significantly increased (4.5 log) in FP when stored under 24 °C and survived at a level of ~105 CFU/g at 10 °C. Comparatively, Sh. sonnie and Sh. flexneri showed a better survival pattern in FP stored under 4 °C and grew (Ë 3 log) after 5 days at 10 and 24 °C. L. monocytogenes was capable of growing by 1.9 and 4.3 log after 14 d days and by 3.9 log after 3 days at 4, 10, or 24 °C, respectively. No significant decline in S. aureus counts at 4 and 10 °C occurred, however, it increased significantly to Ë 7 log CFU/g at 24 °C. Total mesophilic count and yeast and mold count reached to spoilage levels (Ë107 CFU/g) in un-inoculated FP after 1 and 3 days of storage at 24 and 10 °C, respectively. FP could support the growth of common foodborne pathogens and hence it is recommended to utilize natural antimicrobials in FP and keep the product under refrigeration (4 °C) to preclude the growth of vegetative foodborne pathogens.
Subject(s)
Escherichia coli O157 , Listeria monocytogenes , Temperature , Staphylococcus aureus , Food Microbiology , Vegetables , Colony Count, MicrobialABSTRACT
Shigella flexneri is a prevalent foodborne and waterborne pathogen that threatens human health. Our previous research indicated that the Lactiplantibacillus plantarum Y12 exopolysaccharide (L-EPS) potentially inhibited the pathogenicity of S. flexneri. The in vitro results of this study demonstrated that L-EPS effectively mitigated the symptoms induced by S. flexneri in HT-29 cells, including inhibited gene expression levels of IL-1ß, IL-6, IL-8, TNF-α, TLR 2/4, and NOD1/2; decreased apoptosis ratio; and alleviated damage degree of intestinal barrier function (Zona occludens 1, Occludin, and Claudin-1). The in vivo results demonstrated that S. flexneri treated with L-EPS elicited mild adverse physiological manifestations, an inflammatory response, and tissue damage. The infection of S. flexneri caused significant alterations in the abundance of phylum (Firmicutes, Bacteroidota, Actinobacteriota, and Proteobacteria), family (Lachnospiraceae, Muribaculaceae, Rikenellaceae, Prevotellaceaea, Ruminococcaceae, and Lactobaillaceae), and genus (Escherichia Shigella and Lachnospirillaceae NK4A136 group) within the cecal microbiota. These changes were accompanied by perturbations in taurine and hypotaurine metabolism, tricarboxylic acid (TCA) cycle activity, arginine biosynthesis, and histidine metabolic pathways. However, intervention with L-EPS attenuated the dysbiosis of cecal microbiota and metabolic disturbances. In summary, our research suggested a potential application of L-EPS as a functional food additive for mitigating S. flexneri infection.
Subject(s)
Shigella flexneri , Humans , Virulence , HT29 Cells , Biological TransportABSTRACT
The facultative intracellular pathogen Shigella flexneri invades non-phagocytic epithelial gut cells. Through a syringe-like apparatus called type 3 secretion system, it injects effector proteins into the host cell triggering actin rearrangements leading to its uptake within a tight vacuole, termed the bacterial-containing vacuole (BCV). Simultaneously, Shigella induces the formation of large vesicles around the entry site, which we refer to as infection-associated macropinosomes (IAMs). After entry, Shigella ruptures the BCV and escapes into the host cytosol by disassembling the BCV remnants. Previously, IAM formation has been shown to be required for efficient BCV escape, but the molecular events associated with BCV disassembly have remained unclear. To identify host components required for BCV disassembly, we performed a microscopy-based screen to monitor the recruitment of BAR domain-containing proteins, which are a family of host proteins involved in membrane shaping and sensing (e.g. endocytosis and recycling) during Shigella epithelial cell invasion. We identified endosomal recycling BAR protein Sorting Nexin-8 (SNX8) localized to IAMs in a PI(3)P-dependent manner before BCV disassembly. At least two distinct IAM subpopulations around the BCV were found, either being recycled back to cellular compartments such as the plasma membrane or transitioning to become RAB11A positive "contact-IAMs" involved in promoting BCV rupture. The IAM subpopulation duality was marked by the exclusive recruitment of either SNX8 or RAB11A. Hindering PI(3)P production at the IAMs led to an inhibition of SNX8 recruitment at these compartments and delayed both, the step of BCV rupture time and successful BCV disassembly. Finally, siRNA depletion of SNX8 accelerated BCV rupture and unpeeling of BCV remnants, indicating that SNX8 is involved in controlling the timing of the cytosolic release. Overall, our work sheds light on how Shigella establishes its intracellular niche through the subversion of a specific set of IAMs.