Enterohemorrhagic Research Articles

Determining the prevalence of Escherichia coli, Salmonella, and shiga toxin-producing Escherichia coli in manure of dairy lagoons.

The aim of this study was to determine the prevalence of microbial pathogens in manure of dairy lagoons in California. To determine pathogens in dairy manure stored in anaerobic lagoons of dairy farm, an extensive field study was conducted across California to sample manure from 20 dairy farms. Samples were analyzed to determine the prevalence of indicator Escherichia coli, Shiga toxin producing E. coli (STEC), Salmonella, and E. coli O157: H7. To test the E. coli, STEC, and Salmonella, we used agar culture-based method followed by polymerase chain reaction (PCR) method. In addition, a real- time PCR based method was used to determine the presence of E coli O157: H7. Study demonstrated that the prevalence of Salmonella in manure sample is lower than E. coli. The presence of Salmonella was found in 2.26% of the samples, and both the culture-based and PCR methods yielded comparable outcomes in detecting Salmonella. Moreover, ∼11.30% of the total samples out of the 177 were identified as positive for STEC by qPCR. These findings demonstrate that indicator E. coli are abundantly present in anaerobic lagoons. However, the presence of STEC, and Salmonella is substantially low.

Eculizumab for Shiga-toxin-induced hemolytic uremic syndrome in adults with neurological involvement.

The role of eculizumab in treating Shiga-toxin-producing Escherichia coli (STEC) hemolytic uremic syndrome (HUS) patients with neurological involvement remains unclear. We describe two distinctly different STEC-HUS patients with neurologic involvement successfully managed with eculizumab, and perform a literature review of all published cases. Both patients had complete resolution of neurological symptoms after initiation of eculizumab. Eighty patients with STEC-HUS treated with eculizumab were identified in the literature, 68.7% had complete resolution of neurological symptoms. Based on our experience and literature review, three prevailing themes were noted: 1) Early eculizumab administration optimized neurological outcomes, 2) Symptom resolution may not be immediate, neurological symptoms may initially worsen before improvement, and 3) Plasma exchange yielded no benefit. Early administration of eculizumab may reverse neurotoxicity in patients with STEC-HUS.

Comparison of Three Air Sampling Methods for the Quantification of Salmonella, Shiga-toxigenic Escherichia coli (STEC), Coliforms, and Generic E. coli from Bioaerosols of Cattle and Poultry Farms

Recent fresh produce outbreaks potentially associated with bioaerosol contamination from animal operations in adjacent land highlighted the need for further study to better understand the associated risk. The purpose of this research was to evaluate three sampling methods for quantifying target bacterial bioaerosols from animal operations. A dairy cattle and poultry farm located in Georgia, U.S. were visited six times each. Air was collected for 10 min using: 2-stage Andersen impactor with and without mineral oil overlay and impingement samplers. Sampling devices were run concurrently at 0.1, 1, and 2 m heights (n = 36). Andersen samplers were loaded with CHROMagar™ Salmonella, CHROMagar™ STEC, or Brilliance™ coliforms/E. coli. The impingement sampler contained buffered peptone water (20 mL) which was vacuum filtered through a 0.45 µm filter and placed onto the respective media. Plates were incubated at 37 ℃ for 48 h. PCR confirmation followed targeting ttr for Salmonella and stx1, stx2, and eae genes for STEC. No significant differences were found among methods to quantify coliforms and E. coli. Salmonella and STEC bioaerosols were not detected by any of the methods (Limit of detection: 0.55 log CFU/m3). E. coli bioaerosols were significantly greater in the poultry (2.76–5.00 log CFU/m3) than in the cattle farm (0.55–2.82 log CFU/m3) (p < 0.05), and similarly distributed at both stages in the Andersen sampler (stage 1:>7 μm; stage 2: 0.65–7 μm particle size). Sampling day did not have a significant effect on the recovery of coliforms/E. coli bioaerosols in the poultry farm when samples were taken at the broiler house exhaust fan (p > 0.05). A greater and constant emission of coliforms and E. coli bioaerosols from the poultry farm warrants further investigation. These data will help inform bioaerosol sampling techniques which can be used for the quantification of bacterial foodborne pathogens and indicator organisms for future research.

Systemic Effects of a Phage Cocktail on Healthy Weaned Piglets.

Numerous studies have demonstrated that bacteriophages (phages) can effectively treat intestinal bacterial infections. However, research on the impact of phages on overall body health once they enter the intestine is limited. This study utilized weaned piglets as subjects to evaluate the systemic effects of an orally administered phage cocktail on their health. Twelve 21-day-old weaned piglets were divided into control (CON) and phage gavage (Phages) groups. The phage cocktail consisted of five lytic phages, targeting Salmonella enterica serovar Choleraesuis (S. choleraesuis), Enteropathogenic Escherichia coli (EPEC), and Shiga tox-in-producing Escherichia coli (STEC). The phages group received 10 mL of phage cocktail orally for 20 consecutive days. The results show that the phage gavage did not affect the piglets' growth performance, serum biochemical indices, or most organ indices, except for the pancreas. However, the impact on the intestine was complex. Firstly, although the pancreatic index decreased, it did not affect the secretion of digestive enzymes in the intestine. Secondly, phages increased the pH of jejunum chyme and relative weight of the ileum, and enhanced intestinal barrier function without affecting the morphology of the intestine. Thirdly, phages did not proliferate in the intestine, but altered the intestinal microbiota structure and increased concentrations of microbial metabolites isobutyric acid and isovaleric acid in the colonic chyme. In addition, phages impacted the immune status, significantly increasing serum IgA, IgG, and IgM, as well as serum and intestinal mucosal IFN-γ, IL-1β, IL-17, and TGF-β, and decreasing IL-4 and IL-10. They also activated toll-like receptors TLR-4 and TLR-9. Apart from an increase in basophil numbers, the counts of other immune cells in the blood did not change. This study indicates that the impact of phages on body health is complex, especially regarding immune status, warranting further attention. Short-term phage gavage did not have significant negative effects on health but could enhance intestinal barrier function.

Bifidobacterium longum K5 Prevents Enterohaemorrhagic Escherichia coli O157:H7 Infection in Mice through the Modulation of the Gut Microbiota.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a commonly encountered foodborne pathogen that can cause hemorrhagic enteritis and lead to hemolytic uremic syndrome (HUS) in severe cases. Bifidobacterium is a beneficial bacterium that naturally exists in the human gut and plays a vital role in maintaining a healthy balance in the gut microbiota. This study investigated the protective effects of B. longum K5 in a mouse model of EHEC O157:H7 infection. The results indicated that pretreatment with B. longum K5 mitigated the clinical symptoms of EHEC O157:H7 infection and attenuated the increase in myeloperoxidase (MPO) activity in the colon of the mice. In comparison to the model group, elevated serum D-lactic acid concentrations and diamine oxidase (DAO) levels were prevented in the K5-EHEC group of mice. The reduced mRNA expression of tight junction proteins (ZO-1, Occludin, and Claudin-1) and mucin MUC2, as well as the elevated expression of virulence factors Stx1A and Stx2A, was alleviated in the colon of both the K5-PBS and K5-EHEC groups. Additionally, the increase in the inflammatory cytokine levels of TNF-α and IL-1β was inhibited and the production of IL-4 and IL-10 was promoted in the K5-EHEC group compared with the model group. B. longum K5 significantly prevented the reduction in the abundance and diversity of mouse gut microorganisms induced by EHEC O157:H7 infection, including blocking the decrease in the relative abundance of Roseburia, Lactobacillus, and Oscillibacter. Meanwhile, the intervention with B. longum K5 promoted the production of acetic acid and butyric acid in the gut. This study provides insights into the use of B. longum K5 for developing probiotic formulations to prevent intestinal diseases caused by pathogenic bacterial infections.

Foodborne Illnesses from Leafy Greens in the United States: Attribution, Burden, and Cost

Leafy green vegetables are a major source of foodborne illnesses. Nevertheless, few studies have attempted to estimate attribution and burden of illness estimates for leafy greens. This study combines results from three outbreak-based attribution models with illness incidence and economic cost models to develop comprehensive pathogen-specific burden estimates for leafy greens and their subcategories in the United States. We find that up to 9.18% (90% CI: 5.81%-15.18%) of foodborne illnesses linked to identified pathogens are attributed to leafy greens. Including ‘Unknown’ illnesses not linked to specific pathogens, leafy greens account for as many as 2,307,558 (90% CI: 1,077,815–4,075,642) illnesses annually in the United States. The economic cost of these illnesses is estimated to be up to $5.278 billion (90% CI: $3.230-$8.221 billion) annually. Excluding the pathogens with small outbreak sizes, Norovirus, Shiga toxin-producingEscherichia coli (both non-O157 and O157:H7), Campylobacter spp., and nontyphoidal Salmonella, are associated with the highest number of illnesses and greatest costs from leafy greens. While lettuce (romaine, iceberg, “other lettuce”) takes 60.8% of leafy green outbreaks, it accounts for up to 75.7% of leafy green foodborne illnesses and 70% of costs. Finally, we highlighted that 19.8% of Shiga toxin-producingEscherichia coli O157:H7 illnesses are associated with romaine among all food commodities, resulting in 12,496 estimated illnesses and $324.64 million annually in the United States.

Outcome 10years after Shiga toxin-producing E. coli (STEC)-associated hemolytic uremic syndrome: importance of long-term follow-up.

Hemolytic uremic syndrome (HUS) is an important cause of acute kidney injury in children. HUS is known as an acute disease followed by complete recovery, but patients may present with kidney abnormalities after long periods of time. This study evaluates the long-term outcome of Shiga toxin-producing Escherichia coli-associated HUS (STEC-HUS) in pediatric patients, 10years after the acute phase of disease to identify risk factors for long-term sequelae. Over a 6-year period, 619 patients under 18years of age with HUS (490 STEC-positive, 79%) were registered in Austria and Germany. Long-term follow-up data of 138 STEC-HUS-patients were available after 10years for analysis. A total of 66% (n = 91, 95% CI 0.57-0.73) of patients fully recovered showing no sequelae after 10years. An additional 34% (n = 47, 95% CI 0.27-0.43) presented either with decreased glomerular filtration rate (24%), proteinuria (23%), hypertension (17%), or neurological symptoms (3%). Thirty had sequelae 1year after STEC-HUS, and the rest presented abnormalities unprecedented at the 2-year (n = 2), 3-year (n = 3), 5-year (n = 3), or 10-year (n = 9) follow-up. A total of 17 patients (36.2%) without kidney abnormalities at the 1-year follow-up presented with either proteinuria, hypertension, or decreased eGFR in subsequent follow-up visits. Patients needing extracorporeal treatments during the acute phase were at higher risk of presenting symptoms after 10years (p < 0.05). Patients with STEC-HUS should undergo regular follow-up, for a minimum of 10years following their index presentation, due to the risk of long-term sequelae of their disease. An initial critical illness, marked by need of kidney replacement therapy or plasma treatment may help predict poor long-term outcome.

Why put yourself on a pedestal? The pathogenic role of the A/E pedestal.

Certain Escherichia coli (E. coli) strains are attaching and effacing (A/E) lesion pathogens that primarily infect intestinal epithelial cells. They cause actin restructuring and polymerization within the host cell to create an actin-rich protrusion below the site of adherence, termed the pedestal. Although there is clarity on the pathways initiating pedestal formation, the underlying purpose(s) of the pedestal remains ambiguous. The conservation of pedestal-forming activity across multiple pathogens and redundancy in formation pathways indicate a pathogenic advantage. However, few decisive conclusions have been drawn, given that the results vary between model systems. Some research argues that the pedestal increases the colonization capability of the bacterium. These studies utilize A/E pathogens specifically deficient in pedestal formation to evaluate adhesion and intestinal colonization following infection. There have been many proposed mechanisms for the colonization benefit conferred by the pedestal. One suggested benefit is that the pedestal allows for direct cytosolic anchoring through incorporation of the established host cortical actin, causing a stable link between the pathogen and cell structure. The pedestal may confer enhanced motility, as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are better able to migrate on the surface of host cells and infect neighboring cells in the presence of the pedestal. Additionally, some research suggests that the pedestal improves effector delivery. This review will investigate the purpose of pedestal formation using evidence from recent literature and will critically evaluate the methodology and model systems. Most importantly, we will contextualize the proposed functions to reconcile potential synergistic effects.

Using CHROMagar™ STEC medium exclusively does not recover all clinically relevant Shiga toxin-producing Escherichia coli in Aotearoa, New Zealand.

Diagnostic laboratories in Aotearoa, New Zealand (NZ) refer cultures from faecal samples positive for Shiga toxin genes to the national Enteric Reference Laboratory for isolation of Shiga toxin-producing Escherichia coli (STEC) for epidemiological typing. As there was variation in the culture media being referred, a panel of 75 clinical isolates of STEC, representing 28 different serotypes, was used to assess six commercially available media and provide guidance to clinical laboratories. Recommendations were subsequently tested for a 3-month period, where STEC isolations and confirmations were assessed by whole genome sequencing analysis against the culture media referred. CHROMagar™ STEC (CH-STEC; CHROMagar Microbiology, Paris, France) or CH-STEC plus cefixime-tellurite sorbitol MacConkey agar was confirmed inferior to CH-STEC plus blood agar with vancomycin, cefsulodin, and cefixime (BVCC). The former resulted in fewer STEC types (n=18) being confirmed compared to those from a combination of CH-STEC and BVCC (n=42). A significant (P<.05) association with an STEC's ability to grow on CH-STEC and the presence of the ter gene cluster, and eae was observed. Culturing screen positive STEC samples onto both CH-STEC and BVCC ensures a consistently higher recovery of STEC from all clinical samples in NZ than CH-STEC alone.

Molecular detection and antibiogram of Shiga toxin-producing Escherichia coli (STEC) from raw milk in and around Bahir Dar town dairy farms, Ethiopia

Illnesses associated with consuming infected milk and milk products are a widespread problem in low and middle-income countries. Shiga toxin-producing Escherichia coli (STEC) is a bacterium commonly found in raw milk and causes foodborne diseases ranging from mild diarrhea to severe hemorrhagic colitis and hemolytic uremic syndrome. This study aimed to investigate the virulence gene and antimicrobial resistance profiles of Shiga toxin-producing E. coli strains isolated from raw milk in dairy farms in and around Bahir Dar town. Raw milk samples (n = 128) collected from December 2021 to July 2022 were cultured, and E. coli strains were isolated using standard methods. Shiga toxin-producing E. coli strains were identified genotypically by the presence of the virulence markers using a single-plex polymerase chain reaction. The antibiotic susceptibility testing of Shiga toxin-producing E. coli isolates was done by the agar disk diffusion method. In total, 32 E. coli isolates were recovered from milk samples from lactating animals. PCR screening of these isolates resulted in 19 (59.3%) positives for Shiga toxin-producing E. coli. The stx2 gene was detected in 53% of cases, followed by stx1 (31%) and eae (16%. The STEC isolates were highly sensitive to ciprofloxacin (94.7%) and kanamycin (89.5%), while exhibiting significant resistance to amoxicillin (89.5%) and streptomycin (73.7%). The present study points out the occurrence of virulent and antibiotic-resistant Shiga toxin-producing E. coli strains in raw milk that could pose a potential risk to public health. Further analysis by whole genome sequencing is necessary for an in-depth assessment and understanding of their virulence and resistance factors. Moreover, large-scale studies are needed to identify the prevalence and potential risk factors and to prevent the spread of antibiotic-resistant STEC strains in the milk production chain.

Isolation and characterization of a relatively broad-spectrum phage against Escherichia coli.

Multiple pathogenic types or serotypes restrict treatment for colibacillosis. In addition, rising antibiotic resistance has heightened public awareness to prevent and control pathogenic Escherichia coli. The bacteriophage is a viable technique to treat colibacillosis as an alternative to antibiotics. In this study, PH444, a relatively broad-spectrum obligate lytic phage, was screened from 48 Shiga toxin-producing Escherichia coli (STEC) phages isolated from farm manure samples and sewage samples in order to conduct genome-wide analysis, biological characterization, and a bacterial challenge experiment in milk. The results demonstrated that PH444 was a T7-like phage with a double-stranded DNA of 115,111bp that belongs to the Kuravirus and was stable at temperatures between 4 and 50°C and a pH range of 3 to 11. After adding PH444, the bacterial load in milk could be reduced from 3 × 103 PFU/ mL to zero within 1h. In consideration of the biological properties of phage PH444, it was, therefore, demonstrated that PH444 has the potential to be used in phage biocontrol.

Survival of O157 and non-O157 shiga toxin-producing Escherichia coli in Korean style kimchi

Korean style kimchi contaminated with Shiga toxin-producing Escherichia coli (STEC) O157:H7 was the cause of an outbreak in Canada from December 2021 to January 2022. To determine if this STEC O157:H7 has greater potential for survival in kimchi than other STEC, the outbreak strain and six other STEC strains (O26:H11, O91:H21, O103:H2, O121:H19, and two O157:H7) were inoculated individually at 6 to 6.5 log CFU/g into commercially sourced kimchi and incubation at 4 °C. At intervals of seven days inoculated and control kimchi was plated onto MacConkey agar to enumerate lactose utilising bacteria. The colony counts were interpreted as enumerating the inoculated STEC, since no colonies were observed on MacConkey agar plated with uninoculated kimchi. Over eight weeks of incubation the pH was stable at 4.10 to 4.05 and the STEC strains declined by 0.7–1.0 log, with a median reduction of 0.9 log. The linear rate of reduction of kimchi outbreak STEC O157:H7 was −0.4 log per 30 days (Slope Uncertainty 0.05), which was not significantly different from the other O157 and nonO157 STEC strains (P = 0.091). These results indicate that the outbreak was not due to the presence of strain better adapted to survival in kimchi than other STEC, and that STEC can persist in refrigerated Korean style kimchi with a minimal decline over the shelf-life of the product.

A Comprehensive Review on Shiga Toxin Subtypes and Their Niche-Related Distribution Characteristics in Shiga-Toxin-Producing E. coli and Other Bacterial Hosts.

Shiga toxin (Stx), the main virulence factor of Shiga-toxin-producing E. coli (STEC), was first discovered in Shigella dysenteriae strains. While several other bacterial species have since been reported to produce Stx, STEC poses the most significant risk to human health due to its widespread prevalence across various animal hosts that have close contact with human populations. Based on its biochemical and molecular characteristics, Shiga toxin can be grouped into two types, Stx1 and Stx2, among which a variety of variants and subtypes have been identified in various bacteria and host species. Interestingly, the different Stx subtypes appear to vary in their host distribution characteristics and in the severity of diseases that they are associated with. As such, this review provides a comprehensive overview on the bacterial species that have been recorded to possess stx genes to date, with a specific focus on the various Stx subtype variants discovered in STEC, their prevalence in certain host species, and their disease-related characteristics. This review provides a better understanding of the Stx subtypes and highlights the need for rapid and accurate approaches to toxin subtyping for the proper evaluation of the health risks associated with Shiga-toxin-related bacterial food contamination and human infections.

Non-pathogenic Heyndrickxia coagulans (Bacillus coagulans) 29-2E inhibits the virulence of pathogenic Salmonella Typhimurium by quorum-sensing regulation

Bacteria produce and release small signal molecules, autoinducers, as an indicator of their cell density. The system, called a quorum-sensing (QS) system, is used to control not only virulence factors but also antibiotic production, sporulation, competence, and biofilm formation in bacteria. Different from antibiotics, QS inhibitors are expected to specifically repress the virulence factors in pathogenic bacteria without inhibiting growth or bactericidal effects. Therefore, since QS inhibitors have little risk of antibiotic-resistant bacteria emergence, they have been proposed as promising anti-bacterial agents. In the present study, we aimed to find new QS inhibitors that prohibit the signaling cascade of autoinducer 3 (AI-3) recognized by a QseCB two-component system that regulates some virulence factors of pathogens, such as enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica subsp. enterica serovar Typhimurium. We have established the method for QS-inhibitor screening using a newly constructed plasmid pLES-AQSA. E.coli DH5α transformed with the pLES-AQSA can produce β-galactosidase that converts 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal) into blue pigment (5-bromo-4-chloro-indoxyl) under the control of the QseCB system. By screening, Heyndrickxia coagulans (formerly Bacillus coagulans) 29-2E was found to produce an exopolysaccharide (EPS)-like water-soluble polymer that prohibits QseCB-mediated β-galactosidase production without antibacterial activities. Further, the simultaneous injection of the 29-2E strain significantly improves the survival rate of Salmonella Typhimurium-infected silkworm larvae (from 0% to 83.3%), suggesting that the substance may be a promising inhibitor against the virulence of pathogens without risk of the emergence of antibiotic-resistant bacteria.

Structural basis to identify a target site in Shiga toxin for the inhibitor discovery against growth of Shiga toxin-producing E. coli

BackgroundCertain peptides that bind Shiga toxin 2 (Stx2) have been reported to treat Shiga toxin-producing Escherichia coli (STEC) infections. However, their mechanisms of action remain unknown. STEC infections lead to serious diseases, such as hemolytic uremic syndrome, in humans. Antibiotic therapy is usually not recommended because of the major challenges of antibiotic resistance and SOS repair. Currently, there is no human vaccine for STEC infection, leaving rehydration therapy as the recommended supportive therapy. Therefore, there is a need for targeted therapeutic intervention to inhibit STEC growth. The purpose of this study was to evaluate the interaction of five known peptides with Stx2 to identify a more suitable peptide based on structural changes. These peptides have been used to inhibit the growth of STEC.ResultsThe current study demonstrated that only tetravalent peptide (TVP) out of 5 common peptides interrupted the Y77-E259 interaction of Stx2, making it active by exposing active site, which ultimately leads to STEC cell death. We also demonstrated that amino acids R170 and F171 of Stx2 in the docked complex of Stx2 and TVP form a helix-loop-helix (HLH). This might lead to the differential expression of genes regulated by Stx2 and ultimately inhibit STEC growth. However, in the case of Stx2-ribosomal P-stalk, these residues did not form HLH. The 3D refined model of TVP showed a low MolProbity score and low energy zones in the ANOLEA profile compared to the original one. Moreover, the low radius of gyration of the refined TVP suggests that it is more compact than the original TVP. Therefore, TVP is a suitable drug candidate for the inhibition of STEC growth. However, the low antigenicity of TVP makes it unsuitable as a drug candidate. We also evaluated three antibiotics that have been used as active ingredients in FDA-approved peptides. Only Oritavancin diphosphate showed strong polar interactions with Y77-E259 and also had the highest binding affinity.ConclusionsPotential drug candidates that inhibit or interrupt the interaction between Y77-E259 and have high antigenicity, low toxicity, and no allergenicity should be explored against the growth of STEC.

Infection and antibiotic-associated changes in the fecal microbiota of C. rodentium ϕstx2dact-infected C57BL/6 mice.

Enterohemorrhagic Escherichia coli causes watery to bloody diarrhea, which may progress to hemorrhagic colitis and hemolytic-uremic syndrome. While early studies suggested that antibiotic treatment may worsen the pathology of an enterohemorrhagic Escherichia coli (EHEC) infection, recent work has shown that certain non-Shiga toxin-inducing antibiotics avert disease progression. Unfortunately, both intestinal bacterial infections and antibiotic treatment are associated with dysbiosis. This can alleviate colonization resistance, facilitate secondary infections, and potentially lead to more severe illness. To address the consequences in the context of an EHEC infection, we used the established mouse infection model organism Citrobacter rodentium ϕstx2dact and monitored changes in fecal microbiota composition during infection and antibiotic treatment. C. rodentium ϕstx2dact infection resulted in minor changes compared to antibiotic treatment. The infection caused clear alterations in the microbial community, leading mainly to a reduction of Muribaculaceae and a transient increase in Enterobacteriaceae distinct from Citrobacter. Antibiotic treatments of the infection resulted in marked and distinct variations in microbiota composition, diversity, and dispersion. Enrofloxacin and trimethoprim/sulfamethoxazole, which did not prevent Shiga toxin-mediated organ damage, had the least disruptive effects on the intestinal microbiota, while kanamycin and tetracycline, which rapidly cleared the infection without causing organ damage, caused a severe reduction in diversity. Kanamycin treatment resulted in the depletion of all but Bacteroidetes genera, whereas tetracycline effects on Clostridia were less severe. Together, these data highlight the need to address the impact of individual antibiotics in the clinical care of life-threatening infections and consider microbiota-regenerating therapies.IMPORTANCEUnderstanding the impact of antibiotic treatment on EHEC infections is crucial for appropriate clinical care. While discouraged by early studies, recent findings suggest certain antibiotics can impede disease progression. Here, we investigated the impact of individual antibiotics on the fecal microbiota in the context of an established EHEC mouse model using C. rodentium ϕstx2dact. The infection caused significant variations in the microbiota, leading to a transient increase in Enterobacteriaceae distinct from Citrobacter. However, these effects were minor compared to those observed for antibiotic treatments. Indeed, antibiotics that most efficiently cleared the infection also had the most detrimental effect on the fecal microbiota, causing a substantial reduction in microbial diversity. Conversely, antibiotics showing adverse effects or incomplete bacterial clearance had a reduced impact on microbiota composition and diversity. Taken together, our findings emphasize the delicate balance required to weigh the harmful effects of infection and antibiosis in treatment.

RpoS Acts as a Global Repressor of Virulence Gene Expression in Escherichia coli O104:H4 and Enteroaggregative E coli.

In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E coli (EAEC)-specific virulence factors. Here, we identified an E coli O104:H4 isolate that carried a single-nucleotide polymorphism (SNP) in the start codon (ATG > ATA) of rpoS, encoding the alternative sigma factor S. The rpoS ATG > ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E coli O104:H4 Δstx2 and typical EAEC resulted in a similar effect. Both rpoS ATG > ATA and ΔrpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR, encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E coli O104:H4 virulence, primarily through an AggR-dependent mechanism.

A bacterial toxin co-opts caspase-3 to disable active gasdermin D and limit macrophage pyroptosis

During infections, host cells are exposed to pathogen-associated molecular patterns (PAMPs) and virulence factors that stimulate multiple signaling pathways that interact additively, synergistically, or antagonistically. The net effect of such higher-order interactions is a vital determinant of the outcome of host-pathogen interactions. Here, we demonstrate one such complex interplay between bacterial exotoxin- and PAMP-induced innate immune pathways. We show that two caspases activated during enterohemorrhagic Escherichia coli (EHEC) infection by lipopolysaccharide (LPS) and Shiga toxin (Stx) interact in a functionally antagonistic manner; cytosolic LPS-activated caspase-11 cleaves full-length gasdermin D (GSDMD), generating an active pore-forming N-terminal fragment (NT-GSDMD); subsequently, caspase-3 activated by EHEC Stx cleaves the caspase-11-generated NT-GSDMD to render it nonfunctional, thereby inhibiting pyroptosis and interleukin-1β maturation. Bacteria typically subvert inflammasomes by targeting upstream components such as NLR sensors or full-length GSDMD but not active NT-GSDMD. Thus, our findings uncover a distinct immune evasion strategy where a bacterial toxin disables active NT-GSDMD by co-opting caspase-3.

Characteristics of Pathogenic Escherichia coli Associated with Diarrhea in Children under Five Years in Northwestern Ethiopia.

Diarrheagenic Escherichia coli (DEC) are the leading cause of infectious diarrhea and pose a significant global, regional, and national burden of disease. This study aimed to investigate the prevalence of six DEC pathotypes in children with diarrhea and determine their antibiotic resistance patterns. Samples from 107 diarrheagenic children were collected and processed for Escherichia coli (E. coli). Single-plex PCR was used to detect target virulence genes as well as characterize and categorize DEC pathotypes. Antibiotic resistance patterns were determined by the Kirby-Bauer disk diffusion method. E. coli was detected in 79 diarrheal stool samples, accounting for 73.8% of the samples collected. Additionally, 49.4% (39 out of 79) of the isolates harbored various typical virulence factors. Results revealed six pathotypes of virulence: enterotoxigenic E. coli (ETEC) (53.8%), enteropathogenic E. coli (EPEC) (12.8%), enteroaggregative E. coli (EAEC) (10.3%), Heteropathotypes (7.8%), Shiga toxin-producing E. coli (STEC), and enterohemorrhagic E. coli (EHEC) (7.7% each). The isolates exhibited high antibiotic resistance against trimethoprim/sulfamethoxazole (82.1%), amoxicillin (79.5%), ampicillin (74.4%), gentamicin (69.2%), and streptomycin (64.1%). An overall occurrence of 84.6% of multiple-drug resistance was observed in the isolates, with resistance ranging from three to four antibiotic classes. Our findings revealed a high level of pathogenic E. coli that were highly resistant to multiple categories of antibiotics among children in the Awi zone. These findings highlight the potential role of pathogenic E. coli in childhood diarrhea in tropical low-resource settings and underscore the need for continued research on the characteristics of pathogenic and antibiotic-resistant strains.

CobB-mediated deacetylation of the chaperone CesA regulates Escherichia coli O157:H7 virulence

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a common food-borne pathogen that can cause acute diseases. Lysine acetylation is a post-translational modification (PTM) that occurs in various prokaryotes and is regulated by CobB, the only deacetylase found in bacteria. Here, we demonstrated that CobB plays an important role in the virulence of EHEC O157:H7 and that deletion of cobB significantly decreased the intestinal colonization ability of bacteria. Using acetylation proteomic studies, we systematically identified several proteins that could be regulated by CobB in EHEC O157:H7. Among these CobB substrates, we found that acetylation at the K44 site of CesA, a chaperone for the type-III secretion system (T3SS) translocator protein EspA, weakens its binding to EspA, thereby reducing the stability of this virulence factor; this PTM ultimately attenuating the virulence of EHEC O157:H7. Furthermore, we showed that deacetylation of the K44 site, which is deacetylated by CobB, promotes the interaction between CesA and EspA, thereby increasing bacterial virulence in vitro and in animal experiments. In summary, we showed that acetylation influences the virulence of EHEC O157:H7, and uncovered the mechanism by which CobB contributes to bacterial virulence based on the regulation of CesA deacetylation.