Influence of hydrometeorological risk factors on child diarrhea and enteropathogens in rural Bangladesh.

BACKGROUND: A number of studies have detected relationships between weather and diarrhea. Few have investigated associations with specific enteric pathogens. Understanding pathogen-specific relationships with weather is crucial to inform public health in low-resource settings that are especially vulnerable to climate change. OBJECTIVES: Our objectives were to identify weather and environmental risk factors associated with diarrhea and enteropathogen prevalence in young children in rural Bangladesh, a population with high diarrheal disease burden and vulnerability to weather shifts under climate change. METHODS: We matched temperature, precipitation, surface water, and humidity data to observational longitudinal data from a cluster-randomized trial that measured diarrhea and enteropathogen prevalence in children 6 months-5.5 years from 2012-2016. We fit generalized additive mixed models with cubic regression splines and restricted maximum likelihood estimation for smoothing parameters. RESULTS: Comparing weeks with 30°C versus 15°C average temperature, prevalence was 3.5% higher for diarrhea, 7.3% higher for Shiga toxin-producing Escherichia coli (STEC), 17.3% higher for enterotoxigenic E. coli (ETEC), and 8.0% higher for Cryptosporidium. Above-median weekly precipitation (median: 13mm; range: 0-396mm) was associated with 29% higher diarrhea (adjusted prevalence ratio 1.29, 95% CI 1.07, 1.55); higher Cryptosporidium, ETEC, STEC, Shigella, Campylobacter, Aeromonas, and adenovirus 40/41; and lower Giardia, sapovirus, and norovirus prevalence. Other associations were weak or null. DISCUSSION: Higher temperatures and precipitation were associated with higher prevalence of diarrhea and multiple enteropathogens; higher precipitation was associated with lower prevalence of some enteric viruses. Our findings emphasize the heterogeneity of the relationships between hydrometeorological variables and specific enteropathogens, which can be masked when looking at composite measures like all-cause diarrhea. Our results suggest that preventive interventions targeted to reduce enteropathogens just before and during the rainy season may more effectively reduce child diarrhea and enteric pathogen carriage in rural Bangladesh and in settings with similar meteorological characteristics, infrastructure, and enteropathogen transmission.

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.

RNA-Seq analysis of long non-coding RNA in human intestinal epithelial cells infected by Shiga toxin-producing Escherichia coli.

BACKGROUND: The Shiga toxin-producing Escherichia coli (STEC) infects animals and induces acute intestinal inflammation. Long non-coding RNAs (lncRNAs) are known to play crucial roles in modulating inflammation response. However, it is not clear whether lncRNAs are involved in STEC-induced inflammation. METHODS AND RESULTS: To understand the association of lncRNAs with STEC infection, we used RNA-seq technology to analyze the profiles of lncRNAs in Mock-infected and STEC-infected human intestinal epithelial cells (HIECs). We detected a total of 702 lncRNAs differentially expressed by STEC infection. 583 differentially expressed lncRNAs acted as competitive microRNAs (miRNAs) binding elements in regulating the gene expression involved in TNF signaling pathway, IL-17 signaling pathway, PI3K-Akt signaling pathway, and apoptosis pathways. We analyzed 3 targeted genes, TRADD, TRAF1 and TGFB2, which were differentially regulated by mRNA-miRNA-lncRNA interaction network, potentially involved in the inflammatory and apoptotic response to STEC infection. Functional analysis of up/downstream genes associated with differentially expressed lncRNAs revealed their role in adheres junction and endocytosis. We also used the qRT-PCR technique to validate 8 randomly selected differentially expressed lncRNAs and mRNAs in STEC-infected HIECs. CONCLUSION: Our results, for the first time, revealed differentially expressed lncRNAs induced by STEC infection of HIECs. The results will help investigate the molecular mechanisms for the inflammatory responses induced by STEC.

Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified using top-down proteomic analysis.

RATIONALE: Pathogenic bacteria often carry prophage (bacterial viruses) and plasmids (small circular pieces of DNA) that may harbor toxin, antibacterial, and antibiotic resistance genes. Proteomic characterization of pathogenic bacteria should include the identification of host proteins and proteins produced by prophage and plasmid genomes. METHODS: Protein biomarkers of two strains of Shiga toxin-producing Escherichia coli (STEC) were identified using antibiotic induction, matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS) with post-source decay (PSD), top-down proteomic (TDP) analysis, and plasmid sequencing. Alphafold2 was also used to compare predicted in silico structures of the identified proteins to prominent fragment ions generated using MS/MS-PSD. Strain samples were also analyzed with and without chemical reduction treatment to detect the attachment of pendant groups bound by thioester or disulfide bonds. RESULTS: Shiga toxin was detected and/or identified in both STEC strains. For the first time, we also identified the osmotically inducible protein (OsmY) whose sequence unexpectedly had two forms: a full and a truncated sequence. The truncated OsmY terminates in the middle of an α-helix as determined by Alphafold2. A plasmid-encoded colicin immunity protein was also identified with and without attachment of an unidentified cysteine-bound pendant group (~307 Da). Plasmid sequencing confirmed top-down analysis and the identification of a promoter upstream of the immunity gene that is activated by antibiotic induction, that is, SOS box. CONCLUSIONS: TDP analysis, coupled with other techniques (e.g., antibiotic induction, chemical reduction, plasmid sequencing, and in silico protein modeling), is a powerful tool to identify proteins (and their modifications), including prophage- and plasmid-encoded proteins, produced by pathogenic microorganisms.

Shiga toxin targets the podocyte causing hemolytic uremic syndrome through endothelial complement activation.

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) is the leading cause of acute kidney injury in children, with an associated mortality of up to 5%. The mechanisms underlying STEC-HUS and why the glomerular microvasculature is so susceptible to injury following systemic Stx infection are unclear. METHODS: Transgenic mice were engineered to express the Stx receptor (Gb3) exclusively in their kidney podocytes (Pod-Gb3) and challenged with systemic Stx. Human glomerular cell models and kidney biopsies from patients with STEC-HUS were also studied. FINDINGS: Stx-challenged Pod-Gb3 mice developed STEC-HUS. This was mediated by a reduction in podocyte vascular endothelial growth factor A (VEGF-A), which led to loss of glomerular endothelial cell (GEnC) glycocalyx, a reduction in GEnC inhibitory complement factor H binding, and local activation of the complement pathway. Early therapeutic inhibition of the terminal complement pathway with a C5 inhibitor rescued this podocyte-driven, Stx-induced HUS phenotype. CONCLUSIONS: This study potentially explains why systemic Stx exposure targets the glomerulus and supports the early use of terminal complement pathway inhibition in this devastating disease. FUNDING: This work was supported by the UK Medical Research Council (MRC) (grant nos. G0901987 and MR/K010492/1) and Kidney Research UK (grant nos. TF_007_20151127, RP42/2012, and SP/FSGS1/2013). The Mary Lyon Center is part of the MRC Harwell Institute and is funded by the MRC (A410).

Efficacy of Peracetic Acid and Chlorine on the Reduction of Shiga Toxin-producing Escherichia coli and a Nonpathogenic E. coli Strain in Preharvest Agricultural Water.

Produce-borne outbreaks of Shiga toxin-producing Escherichia coli (STEC) linked to preharvest water emphasize the need for efficacious water treatment options. This study quantified reductions of STEC and generic E. coli in preharvest agricultural water using commercially available sanitizers. Water was collected from two sources in Virginia (pond, river) and inoculated with either a seven-strain STEC panel or environmental generic E. coli strain TVS 353 (∼9 log10 CFU/100 mL). Triplicate inoculated water samples were equilibrated to 12 or 32°C and treated with peracetic acid (PAA) or chlorine (Cl) [low (PAA:6ppm, Cl:2-4 ppm) or high (PAA:10 ppm, Cl:10-12 ppm) residual concentrations] for an allotted contact time (1, 5, or 10 min). Strains were enumerated, and a log-linear model was used to characterize how treatment combinations influenced reductions. All Cl treatment combinations achieved a ≥3 log10 CFU/100 mL reduction, regardless of strain (3.43 ± 0.25 to 7.05 ± 0.00 log10 CFU/100 mL). Approximately 80% (19/24) and 67% (16/24) of PAA treatment combinations achieved a ≥3 log10 CFU/100 mL for STEC and E. coli TVS 353, respectively. The log-linear model showed contact time (10 > 5 > 1 min) and sanitizer type (Cl > PAA) had the greatest impact on STEC and E. coli TVS 353 reductions (p < 0.001). E. coli TVS 353 in water samples was more resistant to sanitizer treatment (p < 0.001) indicating applicability as a good surrogate. Results demonstrated Cl and PAA can be effective agricultural water treatment strategies when sanitizer chemistry is managed. These data will assist with the development of in-field validation studies and may identify suitable candidates for the registration of antimicrobial pesticide products for use against foodborne pathogens in preharvest agricultural water treatment.

Detection of hemolytic Shiga toxin-producing Escherichia coli in fresh vegetables and efficiency of phytogenically synthesized silver nanoparticles by Syzygium aromaticum extract and gamma radiation against isolated pathogens.

BACKGROUND: Shiga toxin-producing E. coli (STEC) is a major cause of foodborne diseases accompanied by several clinical illnesses in humans. This research aimed to isolate, identify, and combat STEC using novel alternative treatments, researchers have lately investigated using plant extract to produce nanoparticles in an environmentally acceptable way. At various gamma-ray doses, gamma irradiation is used to optimize the conditions for the biogenically synthesized silver nanoparticles (Ag NPs) using an aqueous extract of clove as a reducing and stabilizing agent. METHODS: On a specific medium, 120 vegetable samples were screened to isolate STEC and molecularly identified using real-time PCR. Moreover, the antibacterial and antibiofilm activities of biogenically synthesized Ag NPs against the isolated STEC were examined. RESULTS: Twenty-five out of 120 samples of eight types of fresh vegetables tested positive for E. coli, as confirmed by 16S rRNA, of which three were positive for the presence of Stx-coding genes, and six were partially hemolytic. Seven antibiotic disks were used to determine antibiotic susceptibility; the results indicated that isolate STX2EC had the highest antibiotic resistance. The results demonstrated that Ag NPs were highly effective against the STEC isolates, particularly the isolate with the highest drug resistance, with inhibition zones recorded as 19 mm for STX2EC, 11 mm for STX1EC1, and 10 mm for STX1EC2 at a concentration of 108 µg/mL. MICs of the isolates STX1EC1, and STX1EC2 were 13.5 µg/mL whereas it was detected as 6.75 µg/mL for STX2EC. The percentages of biofilm inhibition for STX1EC2, STX1EC1, and STX2EC, were 78.7%, 76.9%, and 71.19%, respectively. CONCLUSION: These findings suggest that the biogenic Ag NPs can be utilized as a new promising antibacterial agent to combat biofouling on surfaces.

Expression of hes, iha, and tpsA codified in locus of adhesion and autoaggregation and their involvement in the capability of shiga toxin-producing Escherichia coli strains to adhere to epithelial cells.

OBJECTIVES: Shiga toxin-producing Escherichia coli strains LAA-positive are important cause of human infection. The capability to adhere to epithelial cells is a key virulence trait, and genes codified in LAA pathogenicity island could be involved in the adhesion during the pathogenesis of LAA-positive STEC strains. Thus, our objectives were to compare hes-negative and hes-positive STEC strains in their adherence capability to epithelial cells (HEp-2) and to evaluate the expression levels of the hes, iha, and tpsA in the bacteria adhered and non-adhered to HEp-2 cells. These genes are encoded in LAA, and are virulence factors that participate in adhesion and autoaggregation. RESULTS: We could not observe differences between the adhesion of strains but also in the expression level of of hes, iha, and tpsA. Genes encoded in LAA contribute to the adhesion phenotype though the expression of STEC adhesins is a coordinated event that depends not only the strain but also on the environment as well as its genetic background. Therefore, the results of this study suggest that LAA ,the most prevalent PAI among LEE-negative STEC strains, plays a role in pathogenesis.

Impact of chlorine or peracetic acid on inactivation of Salmonella, Escherichia coli, and Listeria monocytogenes in agricultural water.

Preharvest agricultural water has been recognized as one of the routes of contamination for foodborne pathogens during fruit and vegetable production. Several strategies have been proposed to reduce the risk of pathogens, including preharvest water chemigation, but literature is lacking with regards to microbiological inactivation of common bacterial foodborne pathogens associated with fresh produce contamination, Salmonella enterica, Shiga-toxigenic Escherichia coli (STEC), and Listeria monocytogenes, in surface irrigation water after exposure to chlorine and peracetic acid (PAA). Surface water supplied by a local irrigation district was collected over the summer of 2019. Water was autoclaved, divided into 100 mL samples, and inoculated with a cocktail of five Salmonella, STEC, or Listeria monocytogenes strains or a single strain non-pathogenic E. coli. Samples were then treated with 3, 5, or 7 ppm of free chlorine or PAA, and surviving populations were evaluated using a time-kill assay. A first-order kinetic model was used to fit the inactivation data and obtain the D-values. A secondary model was used to explain the changes due to the type of water, treatment, and microorganism. At a concentration of 3 ppm, the observed and predicted D-values of free chlorine treatments were higher than that of PAA treatments for ground and surface water. Results indicated that PAA was more effective inactivating bacteria than sodium hypochlorite at concentrations of 3 and 5 ppm for both water sources (surface and ground). However, at 7 ppm, the effectiveness of PAA and sodium hypochlorite showed no statistically significant difference for both surface and groundwater. Findings will provide information regarding efficacy of chemical sanitizers like chlorine and PAA for inactivation of Salmonella, Listeria, and STEC in surface water from which treatments can be derived. Ultimately benefitting growers in the selection of an appropriate method for in-field treatment of irrigation water if deemed necessary.

Bruton's tyrosine kinase inhibition attenuates disease progression by reducing renal immune cell invasion in mice with hemolytic-uremic syndrome.

Hemolytic-uremic syndrome (HUS) can occur as a complication of an infection with Shiga-toxin (Stx)-producing Escherichia coli. Patients typically present with acute kidney injury, microangiopathic hemolytic anemia and thrombocytopenia. There is evidence that Stx-induced renal damage propagates a pro-inflammatory response. To date, therapy is limited to organ-supportive strategies. Bruton's tyrosine kinase (BTK) plays a pivotal role in recruitment and function of immune cells and its inhibition was recently shown to improve renal function in experimental sepsis and lupus nephritis. We hypothesized that attenuating the evoked immune response by BTK-inhibitors (BTKi) ameliorates outcome in HUS. We investigated the effect of daily oral administration of the BTKi ibrutinib (30 mg/kg) and acalabrutinib (3 mg/kg) in mice with Stx-induced HUS at day 7. After BTKi administration, we observed attenuated disease progression in mice with HUS. These findings were associated with less BTK and downstream phospholipase-C-gamma-2 activation in the spleen and, subsequently, a reduced renal invasion of BTK-positive cells including neutrophils. Only ibrutinib treatment diminished renal invasion of macrophages, improved acute kidney injury and dysfunction (plasma levels of NGAL and urea) and reduced hemolysis (plasma levels of bilirubin and LDH activity). In conclusion, we report here for the first time that BTK inhibition attenuates the course of disease in murine HUS. We suggest that the observed reduction of renal immune cell invasion contributes - at least in part - to this effect. Further translational studies are needed to evaluate BTK as a potential target for HUS therapy to overcome currently limited treatment options.

Genomic diversity of antimicrobial-resistant and Shiga toxin gene-harboring non-O157 Escherichia coli from dairy calves.

OBJECTIVES: Shiga toxin-producing Escherichia coli (STEC) are globally significant foodborne pathogens. Dairy calves are a known reservoir of both O157 and non-O157 STEC. The objective of this study was to comprehensively evaluate the genomic attributes, diversity, virulence factors, and antimicrobial resistance gene (ARG) profiles of the STEC from preweaned and postweaned dairy calves in commercial dairy herds. METHODS: In total, 31 non-O157 STEC were identified as part of a larger study focused on the pangenome of >1000 E. coli isolates from the faeces of preweaned and postweaned dairy calves on commercial dairy farms. These 31 genomes were sequenced on an Illumina NextSeq500 platform. RESULTS: Based on the phylogenetic analyses, the STEC isolates were determined to be polyphyletic, with at least three phylogroups: A (32%), B1 (58%), and G (3%). These phylogroups represented at least 16 sequence types and 11 serogroups, including two of the 'big six' serogroups, O103 and O111. Several Shiga toxin gene subtypes were identified in the genomes, including stx1a, stx2a, stx2c, stx2d, and stx2g. Using the ResFinder database, the majority of the isolates (>50%) were determined to be multidrug-resistant strains because they harboured genes conferring resistance to three or more classes of antimicrobials, including some of human health significance (e.g., ß-lactams, macrolides, and fosfomycin). Additionally, non-O157 STEC strain persistence and transmission within a farm was observed. CONCLUSION: Dairy calves are a reservoir of phylogenomically diverse multidrug-resistant non-O157 STEC. Information from this study may inform assessments of public health risk and guide preharvest prevention strategies focusing on STEC reservoirs.

Effect of potassium tellurite concentration in a chromogenic agar medium on isolation of tellurite-resistant "Top Seven" Shiga toxin-producing Escherichia coli from ground beef.

The effect of potassium tellurite concentration in a chromogenic agar medium on the detection of tellurite-resistant "top seven" Shiga toxin-producing Escherichia coli (STEC) in beef was evaluated. Samples of ground beef were inoculated with tellurite-resistant STEC O26, O45, O103, O111, O121, O145, or O157 strains at geometric mean (±standard error of the mean) levels of 0, 49 (±1), 490 (±1), or 4900 (±1) CFU/10 g and enriched 1:10 (90 mL) in EC broth (40°C for 6 h). Following enrichment, aliquots of broth culture were treated by immunomagnetic separation with one of three pools of beads against STEC serogroups; pool I: O26, O45, and O121; pool II: O103, O111, and O145; and pool III: O157. After immunomagnetic separation, 50 µL of washed bead suspensions in buffered peptone water was spiral plated onto a modified Possé medium containing 0.5, 1.0, or 1.5 mg/L potassium tellurite, and incubated at 37°C for 18 h. Up to four isolated colonies were picked from each spiral plate based on expected colony phenotypes for STEC, and isolate identity was confirmed with an 11-plex PCR assay targeting the O serogroups and virulence genes. Overall, across all inoculum levels and strains, modified Possé media containing 0.5, 1.0, or 1.5 mg/L potassium tellurite each had a positive predictive value of 100%, and medium containing 0.5 mg/L potassium tellurite had numerically the highest sensitivity (100%) and negative predictive value (100%), which was significantly different from 1.5 mg/L (92.9% and 40.0%, respectively; P < 0.05). Similarly, there was an inverse relationship between potassium tellurite concentration and analytical specificity (number of colonies tested that were STEC-positive): 0.5 (1463 of 1482; 98.7%), 1.0 (1356 of 1411; 96.1%), and 1.5 mg/L (1187 of 1278; 92.9%; P < 0.05). These results suggest that 0.5 mg/L gives better performance than 1.0 or 1.5 mg/L of potassium tellurite in Possé medium for isolation of tellurite-resistant "top seven" STEC from ground beef.

Growth behavior of Shiga toxin-producing Escherichia coli, Salmonella, and generic E. coli in raw pork considering background microbiota at 10, 25, and 40 °C.

Recent epidemiological evidence suggests that pork products may be vehicles for the transmission of Shiga toxin-producing Escherichia coli (STEC) to humans. The severe morbidity associated with STEC infections highlights the need for research to understand the growth behavior of these bacteria in pork products. Classical predictive models can estimate pathogen growth in sterile meat. However, competition models considering background microbiota reflect a more realistic scenario for raw meat products. The objective of this study was to estimate the growth kinetics of clinically significant STEC (O157, non-O157, and O91), Salmonella, and generic E. coli in raw ground pork using competition primary growth models at temperature abuse (10 and 25 °C) and sublethal temperature (40 °C). A competition model incorporating the No lag Buchanan model was validated using the acceptable prediction zone (APZ) method where >92 % (1498/1620) of the residual errors fell within the APZ (pAPZ > 0.70). The background microbiota (mesophilic aerobic plate counts, APC) inhibited the growth of STEC and Salmonella indicating a simple one-directional competitive interaction between pathogens and the mesophilic microbiota of ground pork. The maximum specific growth rate (µmax) of all the bacterial groups was not significantly different (p > 0.05) based on fat content (5 vs 25 %) except for generic E. coli at 10 °C. E. coli O157 and non-O157 behaved similarly in terms of µmax and maximum population density (MPD). Salmonella showed a similar (p > 0.05) µmax to E. coli O157 and non-O157 at 10 and 40 °C but a significantly higher rate (p < 0.05) at 25 °C. STEC were more prone to be inhibited by APC than Salmonella at 10 and 25 °C. The µmax of O91 was lower (p < 0.05) than other STEC and Salmonella at 10 and 25 °C but similar (p > 0.05) at 40 °C. Generic E. coli showed a two- to five-times higher (p < 0.05) µmax (0.028 ± 0.011 log10 CFU/h) than other bacterial groups (0.006 ± 0.004 to 0.012 ± 0.003 log10 CFU/h) at 10 °C making it a potential indicator bacteria for process control. Industry and regulators can use competitive models to develop appropriate risk assessment and mitigation strategies to improve the microbiological safety of raw pork products.

Haemolytic Uremic Syndrome: A Study Cohort over 10-Year Period in a Paediatric Tertiary Centre Hospital.

BACKGROUND: Haemolytic uremic syndrome (HUS) is a thrombotic microangiopathy characterized by haemolytic anaemia, thrombocytopenia, and acute kidney injury. It represents the most frequent cause of acute kidney failure in paediatric age. HUS includes acquired types, such as post-infectious forms, and inherited types. If not promptly recognized, HUS still has high mortality and morbidity, with disabling long-term sequelae. METHODS: Children diagnosed with HUS hospitalized between January 2010 and July 2021 at Meyer Children's Hospital were retrospectively studied. RESULTS: We selected 33 patients (M:F = 15:18) with a median age of 40 months (range 12-180 months). Twenty-eight cases (84.8%) were classified as acquired HUS: Shiga-like toxin Escherichia coli-related-HUS (STEC-HUS) was diagnosed in 26 patients (78.8%), while other 2 patients had HUS secondary to Streptococcus pneumoniae infections (3%) and hematopoietic stem cell transplantation (3%), each one. Five cases (15.1%) were classified as hereditary HUS: 4 patients (12.1%) presented inherited complement disorders (atypical HUS); 1 patient (3%) was diagnosed with cobalamin C deficiency. Diarrhoea was the most rated symptom (72.7%), mainly in STEC-HUS forms. In hereditary HUS, kidney involvement manifestations prevailed. Hypertension was present in 54.5% of total cases. Hypocomplementemia was present in 48.5% of patients; 30.3% of patients needed hospitalization in paediatric intensive care unit (PICU). Early hypertension and hypocomplementemia resulted to be related to the disease severity for either acute phase or long-term outcome. Leucocytosis, thrombocytopenia, and worsen renal function indices were related to PICU hospitalization. Overall, the outcome was good: long-term complications persisted in 18.2% of cases; 1 patient developed kidney failure; no patient died. CONCLUSIONS: HUS is a multifactorial disease mostly affecting children between 3 and 5 years old. Hypertension, leucocytosis, hypocomplementemia, thrombocytopenia, increased renal function indices, and extrarenal manifestations are risk factors for the worst outcome.

Targeting the innate repair receptor axis via erythropoietin or pyroglutamate helix B surface peptide attenuates hemolytic-uremic syndrome in mice.

Hemolytic-uremic syndrome (HUS) can occur as a systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli and is characterized by microangiopathic hemolytic anemia and acute kidney injury. Hitherto, therapy has been limited to organ-supportive strategies. Erythropoietin (EPO) stimulates erythropoiesis and is approved for the treatment of certain forms of anemia, but not for HUS-associated hemolytic anemia. EPO and its non-hematopoietic analog pyroglutamate helix B surface peptide (pHBSP) have been shown to mediate tissue protection via an innate repair receptor (IRR) that is pharmacologically distinct from the erythropoiesis-mediating receptor (EPO-R). Here, we investigated the changes in endogenous EPO levels in patients with HUS and in piglets and mice subjected to preclinical HUS models. We found that endogenous EPO was elevated in plasma of humans, piglets, and mice with HUS, regardless of species and degree of anemia, suggesting that EPO signaling plays a role in HUS pathology. Therefore, we aimed to examine the therapeutic potential of EPO and pHBSP in mice with Stx-induced HUS. Administration of EPO or pHBSP improved 7-day survival and attenuated renal oxidative stress but did not significantly reduce renal dysfunction and injury in the employed model. pHBSP, but not EPO, attenuated renal nitrosative stress and reduced tubular dedifferentiation. In conclusion, targeting the EPO-R/IRR axis reduced mortality and renal oxidative stress in murine HUS without occurrence of thromboembolic complications or other adverse side effects. We therefore suggest that repurposing EPO for the treatment of patients with hemolytic anemia in HUS should be systematically investigated in future clinical trials.

Outbreak of hemolytic uremic syndrome with unusually severe clinical presentation caused by Shiga toxin-producing Escherichia coli O26:H11 in France.

BACKGROUND: In spring 2019, an outbreak of Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC HUS) occurred in France. Epidemiological investigations made by Santé publique France in connection with microbiological investigations at the national reference center for STEC promptly identified a common exposure to consumption of raw cow's milk cheese, and confirmed a cluster affiliation of the E. coli O26:H11 outbreak strain. Here, we report the clinical characteristics of the patients, the treatment used, as well as the outcome at 1 month. METHOD: Patients with STEC HUS linked to the E. coli O26:H11 outbreak strain were identified from the national surveillance network of pediatric STEC HUS cases coordinated by Santé publique France. Clinical data were analyzed from the patients' hospital records obtained from the treating physicians. RESULTS: Overall, 20 pediatric cases of STEC HUS linked to the outbreak strain were identified. Their median age of the patients was 16 months (range: 5-60). Most of them presented with diarrhea but none had received prior antibiotherapy. A total of 13 patients required dialysis; 10 patients and four patients had central nervous system (CNS) and cardiac involvement, respectively. No deaths occurred. At the 1-month follow-up, only two patients had a decreased glomerular filtration rate, below 80 mL /min/1.73m2 and four had hypertension. One patient had neurological sequelae. CONCLUSION: The E. coli O26:H11 strain identified as the cause of an STEC HUS outbreak in France in spring 2019 is notable for the initial severe clinical presentation of the patients, with a particularly high frequency of CNS and cardiac involvement similar to the German E. coli O104:H4 outbreak described in 2011. However, despite the initial severity, the 1-month outcome was favorable in most cases. The patients' young age in this outbreak highlights the need to improve information and caregiver awareness regarding consumption of at-risk foods by young children as key preventive measures against STEC infections.

Caracterización genotípica y biofilms de E. coli shigatoxigénica aisladas de casos clínicos humanos / Genotypic characterization and biofilms of shigatoxigenic E. coli isolated from human clinical cases

Resumen Escherichia coli shigatoxigénica (STEC) está involucrada en el desarrollo del síndrome urémico hemolítico, entre otras enfermedades que son de gran importancia para la salud pública e inocuidad alimentaria a nivel mundial. La capacidad de STEC de formar biofilms en los alimentos y en diferentes superficies podría conducir a la contaminación cruzada por el desprendimiento de las células bacterianas. El objetivo del presente trabajo fue detectar la presencia de genes que codifican factores de adherencia mediante la técnica de PCR y determinar la capacidad de formación de biofilms por medio de cultivo en microplacas de poliestireno de 96 pocillos y la técnica de cristal violeta, en cepas de STEC aisladas de muestras clínicas humanas en la ciudad de Mar del Plata, Argentina. El perfil de genes de adherencia más frecuente fue efa1, iha, fimCD, ehaA, lpfA1-3, lpfA2-2, cah (43,9%). Todas las cepas de STEC formaron biofilms con valores de densidad óptica entre 0,209 y 3,251 y el 54,4% (31/57) de las mismas fueron clasificadas como fuertes formadoras de biofilms. La capacidad de formación de biofilms de STEC constituye un riesgo evidente en la transmisión de este patógeno al ser humano a tener en cuenta para su vigilancia y control.

Abstract Shigatoxigenic Escherichia coli (STEC) is involved in the development of hemolytic uremic syndrome, among other diseases that are relevant to public health and food safety worldwide. The ability of STEC to form biofilms in food and on different surfaces could lead to cross-contamination by shedding bacterial cells. The aim of this work was to detect the presence of genes encoding adherence factors by the PCR technique and to determine the biofilm formation ability by culture in 96-well polystyrene microplates and the crystal violet technique, in STEC strains isolated from human clinical samples in Mar del Plata city, Argentina. The most frequent adherence gene profile was efa1, iha, fimCD, ehaA, lpfA1-3, lpfA2-2, cah (43.9%). All STEC strains formed biofilms with optical density values between 0.209 and 3.251. Also, the 54.4% (31/57) of STEC strains were classified as strong biofilm formers. The ability of STEC to form biofilms constitutes an evident risk in the transmission of this pathogen to humans, which must be taken into account for its surveillance and control.

Resumo A Escherichia coli shigatoxigênica (STEC) está envolvida no desenvolvimento da síndrome hemolítica urêmica, entre outras doenças relevantes para a saúde pública e segurança alimentar em todo o mundo. A capacidade do STEC de formar biofilmes nos alimentos e em diferentes superfícies poderia levar à contaminação cruzada através do desprendimento de células bacterianas. O objetivo do presente trabalho foi detectar a presença de genes que codificam fatores de aderência através da técnica PCR e determinar a capacidade de formação de biofilme por cultura em microplacas de poliestireno de 96 poços e da técnica de cristal violeta, em cepas STEC isoladas de amostras clínicas humanas na cidade de Mar del Plata, Argentina. O perfil de genes de aderência mais frequente foi efa1, iha, fimCD, ehaA, lpfA1-3, lpfA2-2, cah (43,9%). Todas as cepas de STEC formaram biofilmes com valores de densidade ótica entre 0,209 e 3,251. Também, os 54,4% (31/57) das estirpes STEC foram classificados como fortes formadores de biofilmes. A habilidade de formação de biofilmes de STEC constitui um risco evidente na transmissão deste patógeno ao humano, que deve ser levado em consideração para sua vigilância e controle.

Isolation of a multidrug-resistant Escherichia coli pathotype Stx2:Cnf1:Cnf2:Eae as a potential cause of hemorrhagic diarrhea and secondary septicemia in a dog.

Escherichia coli is a member of the family Enterobacteriaceae and is a commensal in the intestine of many animals as well as humans. Most strains are of low virulence. A dog developed vomiting and hemorrhagic diarrhea after surgery and died despite treatment. Postmortem examination revealed hemorrhagic gastroenteritis and colitis. A multidrug-resistant E. coli, with virulence factors Shiga-toxin-producing gene, stx2, eae gene, and cytotoxic necrotic factors CNF-1 and CNF-2, was isolated from internal organs. E. coli can easily acquire new genes for virulence factors and antimicrobial resistance as demonstrated by this isolate with characteristics of both enterohemorrhagic E. coli and necrotoxigenic E. coli. In addition, the isolate was resistant to all beta-lactam antibiotics tested, as well as to enrofloxacin by a disk diffusion methodology. Broth-based minimum inhibitory concentration analysis confirmed resistance to amoxicillin (>32 µg/mL), enrofloxacin (>32 µg/mL), fosfomycin (>128 µg/mL), and neomycin (>32 µg/mL). The discovery of such strains is a cause for concern given that E. coli can be shared by companion animals and their human owners.

Growth medium- and strain-dependent bactericidal efficacy of blue light against Shiga toxin-producing Escherichia coli on food-grade stainless steel and plastic.

Shiga toxin-producing Escherichia coli (STEC) are a major group of human pathogens and may persist on both abiotic and biotic surfaces. In this report, two blue-light prototypes were used to evaluate the antimicrobial efficacy against STEC on food processing surfaces (stainless steel and polyoxymethylene plastic). Investigation using a light-bulb prototype (Prototype 1 at 405 nm, 26 mW/cm2) showed significant antimicrobial effects in nutrient deficient condition but not in nutrient rich condition, demonstrating that the presence of organic matters from rich nutrient medium was thought to be light-absorptive and reduce the bactericidal efficacy of blue light, as evident from the lack of bacterial reduction when suspended in cooked meat broth. An advanced (surface-mounted-diode) light panel, Prototype 2 with high light intensity (405 nm; 50 mW/cm2) was able to inactivate a cocktail of seven STEC strains (from seven major serotypes O26, O45, O103, O111, O121, O145 and O157) on type 304 stainless steel (1.66 log10 CFU) and polyoxymethylene plastic (4.25 log10 CFU) at light dosages of 720 and 45 J/cm2, respectively when cells were illuminated in a nutrient-deficient medium (M9 broth). Post-treatment, no STEC cells were recoverable from plastic, both when tested on plates (agar or petrifilms) and by polymerase chain reaction (PCR). In contrast, surviving colonies were identified on samples taken from stainless steel, albeit only four strains could be detected by PCR analysis - those belonging to serotypes O26, O45, O103 and O157 - which indicated that the susceptibility of STEC to blue light varied across the tested strains.