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Enterococcal bloodstream infections (EBSIs) are rare infections in children associated with 5%-10% of mortality in previous studies. The recent evolution of antimicrobial resistance and therapies require updated data.We conducted an observational retrospective study between January 2008 and December 2019 describing the characteristics of children with EBSI in a French pediatric hospital. All positive Enterococcus spp. blood cultures associated with sepsis symptoms were analyzed. We also compared characteristics of healthcare-associated infections (HAIs) and community-acquired infections (CAIs) and described antimicrobial resistance evolution during this period.In total 74 EBSI were included. Enterococcus faecalis was the most common pathogen (n = 60/74, 81%) followed by Enterococcus faecium (n = 18, 24%), including 4 enterococcal coinfections. EBSIs were mainly associated with central-line associated infection (38%), surgical site infection (14%) or urinary tract infection (11%). An underlying disease was present in 95.9%. However, 4 patients died in the month following the EBSI resulting in a 5.4%, 30-day mortality. All were HAI. HAI (84% of EBSI) was associated with longer bacteremia [31% persistent bacteremia (more than 3 days) versus 0% for CAI; P = 0.029] and more antimicrobial resistance. Amoxicillin resistance is increasing since 2013 in E. faecium (63% in 2013-2019), although high-level gentamicin resistance is stable (19%). Only 1 EBSI due to vancomycin-resistant Enterococcus was described in our cohort, who died.EBSIs are rare infections in children mostly described in children with underlying disease. Healthcare-associated bacteremia is associated with higher rates of resistance and poorer prognosis, requiring the involvement of pediatric infectious disease specialists to improve management.

ObjectivesMultiplex gastrointestinal PCR (GI-PCR) allows fast and simultaneous detection of 22 enteric pathogens (including Campylobacter, Salmonella, Shigella/enteroinvasive Escherichia coli (EIEC), among other bacteria, parasites and viruses). However, its impact on the management of children with infectious diarrhoea remains unknown.Patients/DesignAll children eligible for stool culture from May to October 2018 were prospectively included in a monocentric study at Robert-Debré University-Hospital.InterventionA GI-PCR (BioFire FilmArray) was performed on each stool sample.Main measuresData on the children’s healthcare management before and after GI-PCR results were collected. Stool culture results were also reported.Results172 children were included. The main criteria for performing stool analysis were mucous/bloody diarrhoea and/or traveller’s diarrhoea (n=130). GI-PCR’s were positive for 120 patients (70%). The main pathogens were enteroaggregative E. coli (n=39; 23%), enteropathogenic E. coli (n=34; 20%), Shigella/EIEC (n=27; 16%) and Campylobacter (n=21; 12%). Compared with stool cultures, GI-PCR enabled the detection of 21 vs 19 Campylobacter, 12 vs 10 Salmonella, 27 Shigella/EIEC vs 13 Shigella, 2 vs 2 Yersinia enterocolitica, 1 vs 1 Plesiomonas shigelloides, respectively. Considering the GI-PCR results and before stool culture results, the medical management was revised for 40 patients (23%): 28 initiations, 2 changes and 1 discontinuation of antibiotics, 1 hospitalisation, 2 specific room isolations related to Clostridioides difficile infections, 4 additional test prescriptions and 2 test cancellations.ConclusionThe GI-PCR’s results impacted the medical management of gastroenteritis for almostone-fourth of the children, and especially the prescription of appropriate antibiotic treatment before stool culture results.

We conducted a retrospective study on hemolytic uremic syndrome caused by Shiga toxin-producing Escherichia coli (STEC) in 96 adults enrolled in the cohort of the National Reference Center for Thrombotic Microangiopathies network in France during 2009-2017. Most infections were caused by STEC strains not belonging to the O157 or O104 serogroups. Thirty (31.3%) patients had multiple risk factors for thrombotic microangiopathy. In total, 61 (63.5%) patients required dialysis, 50 (52.1%) had a serious neurologic complication, 34 (35.4%) required mechanical ventilation, and 19 (19.8%) died during hospitalization. We used multivariate analysis to determine that the greatest risk factors for death were underlying immunodeficiency (hazard ratio 3.54) and severe neurologic events (hazard ratio 3.40). According to multivariate analysis and propensity score-matching, eculizumab treatment was not associated with survival. We found that underlying conditions, especially immunodeficiency, are strongly associated with decreased survival in adults who have hemolytic uremic syndrome caused by STEC.

BackgroundAcute chest syndromes (ACS) may be associated with upper respiratory tract infections, but the epidemiology of viral and intracellular respiratory pathogens in children with sickle cell disease (SCD) is not precisely known. The aim of this study was to describe the epidemiology of viral and intracellular respiratory pathogens in children with SCD presenting with fever and/or ACS.Materials and methodsAn observational, prospective, single-centre cohort study with nested case–control analysis was conducted on children with SCD admitted from October 2016 to October 2017 for fever and/or ACS to the paediatric department of Robert Debré university hospital, Paris, France. They were screened for 20 respiratory pathogens by a multiplex PCR in the nasopharynx (FilmArray).ResultsWe included 101 children. M/F sex ratio of 0.45. The median age was 3.2 years (IQR: 1.4–8.2). At least one pathogen was isolated in 67 patients (67%). The most frequent viruses were as follows: rhinovirus (n=33), adenovirus (n=14), respiratory syncytial virus (n=13) and parainfluenza viruses (n=11). Mycoplasma pneumoniae was detected in one case. Twenty-three (23%) presented with or developed ACS. A nested case–control analysis was performed, after pairing ACS with non-ACS children for age and inclusion period. There was no statistical association between any viral detection or multiple viral infection, and ACS (p=0.51) even though parainfluenza viruses were twice as common in ACS.ConclusionsViral detection in febrile children with SCD is frequent, but its association with ACS was not demonstrated. In this study, M. pneumoniae was rare in young children with SCD experiencing ACS.

We described and characterized Shiga-toxin-producing Escherichia coli (STEC) strains with high levels of resistance to azithromycin isolated in France between 2004 and 2020. Nine of 1,715 (0.52%) STEC strains were resistant to azithromycin, with an increase since 2017. One isolate carried a plasmid-borne mef(C)-mph(G) gene combination, described here for the first time for E. coli. Azithromycin resistance, although rare, needs consideration, as this treatment may be useful in cases of STEC infection.

Enterohemorrhagic Escherichia coli (EHEC) O80:H2, belonging to sequence type ST301, is among the main causes of hemolytic and uremic syndrome in Europe, a major concern in young children. Aside from the usual intimin and Shiga toxin virulence factors (VFs), this emerging serotype possesses a mosaic plasmid combining extra-intestinal VF- and antibiotic resistance-encoding genes. This hybrid pathotype can be involved in invasive infections, a rare occurrence in EHEC infections. Here, we aimed to optimize its detection, improve its clinical diagnosis, and identify its currently unknown reservoir. O80:H2 EHEC strains isolated in France between 2010 and 2018 were phenotypically and genetically analyzed and compared with non-O80 strains. The specificity and sensitivity of a PCR test and a culture medium designed, based on the molecular and phenotypic signatures of O80:H2 EHEC, were assessed on a collection of strains and stool samples. O80:H2 biotype analysis showed that none of the strains (n = 137) fermented melibiose versus 5% of non-O80 EHEC (n = 19/352). This loss of metabolic function is due to deletion of the entire melibiose operon associated with the insertion of a 70-pb sequence (70mel), a genetic scar shared by all ST301 strains. This metabolic hallmark was used to develop a real-time PCR test (100% sensitivity, 98.3% specificity) and a melibiose-based culture medium including antibiotics, characterized by 85% specificity and sensitivity for clinical specimens. These new tools may facilitate the diagnosis of this atypical clone, help the food industry to identify the reservoir and improve our epidemiological knowledge of this threatening and emerging clone.

International audience; The severity of human infection by one of the many Shiga toxin-producing Escherichia coli (STEC) is determined by a number of factors: the bacterial genome, the capacity of human societies to prevent foodborne epidemics, the medical condition of infected patients (in particular their hydration status, often compromised by severe diarrhea), and by our capacity to devise new therapeutic approaches, most specifically to combat the bacterial virulence factors, as opposed to our current strategies that essentially aim to palliate organ deficiencies. The last major outbreak in 2011 in Germany, which killed more than 50 people in Europe, was evidence that an effective treatment was still lacking. Herein, we review the current knowledge of STEC virulence, how societies organize the prevention of human disease, and how physicians treat (and, hopefully, will treat) its potentially fatal complications. In particular, we focus on STEC-induced hemolytic and uremic syndrome (HUS), where the intrusion of toxins inside endothelial cells results in massive cell death, activation of the coagulation within capillaries, and eventually organ failure. Key Contribution: We review here the current understanding of the mechanisms of virulence of Shiga toxin-producing Escherichia coli; in particular how infection can lead to thrombotic microangiopathy and acute kidney injury. A modern procedure for differential diagnosis is also provided.

OBJECTIVES Thrombotic microangiopathy syndromes are a heterogeneous group of severe diseases that often require ICU admission. Prompt initiation of targeted therapies is required for atypical hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, whereas there is no specific consensus therapy for Shiga toxin-associated hemolytic uremic syndrome. We sought to compare the characteristics of Shiga toxin-associated hemolytic uremic syndrome, atypical hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura patients at admission in the ICU to allow early differentiation of Shiga toxin-associated hemolytic uremic syndrome from other thrombotic microangiopathy syndromes and help to tailor initial treatment. DESIGN Retrospective cohort study. SETTING Two ICUs part of the French reference center for thrombotic microangiopathy syndromes. PATIENTS Adult patients presenting with features of thrombotic microangiopathy syndromes. Other causes than Shiga toxin-associated hemolytic uremic syndrome, atypical hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura were excluded. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS From September 2003 to January 2017, 236 thrombotic microangiopathy syndrome patients were admitted, including 12 Shiga toxin-associated hemolytic uremic syndrome, 21 atypical hemolytic uremic syndrome, and 91 thrombotic thrombocytopenic purpura. Shiga toxin-associated hemolytic uremic syndrome patients were older than other thrombotic microangiopathy syndromes patients (64 yr [interquartile range, 50-72 yr] vs 42 yr [31-54 yr]; p = 0.007) and presented with more frequent digestive symptoms (92% vs 42%; p < 0.001), especially nonbloody diarrhea and vomiting. Biologically, Shiga toxin-associated hemolytic uremic syndrome patients displayed higher fibrinogen (490 mg/dL [460-540 mg/dL] vs 320 mg/dL [240-410 mg/dL]; p = 0.003) and creatinine levels (2.59 mg/dL [2.12-3.42 mg/dL] vs 1.26 mg/dL [0.61-1.90 mg/dL]; p < 0.001), and less marked anemia (hemoglobin level, 9.7 g/dL [8.7-11.9 g/dL] vs 7.7 g/dL [6.3-9.1 g/dL]; p < 0.001). Forty-two percent (n = 5) required renal replacement therapy, and 83% (n = 10) were treated with plasma exchange before the distinction from other thrombotic microangiopathy syndromes could be made. CONCLUSIONS Adult Shiga toxin-associated hemolytic uremic syndrome patients are older, present more frequently with digestive symptoms and display higher hemoglobin and fibrinogen levels than other thrombotic microangiopathy syndromes. However, overlap across the three thrombotic microangiopathy syndromes remains substantial, putting forward the need to implement early plasma therapy until thrombotic thrombocytopenic purpura and atypical hemolytic uremic syndrome can be ruled out.

International audience

International audience; Invasive meningococcal disease (IMD) is usually associated with intense inflammatory response that is correlated with severe infection. Corticosteroids may regulate this inflammatory response through an early but transient induction of IL-10 that is suggested to improve the outcome of IMD. We explored the mechanism of action of corticosteroids as an adjuvant treatment to antibiotics. Transgenic mice expressing the human transferrin were infected by a hyperinvasive meningococcal strain and transcriptomic analysis were then performed in the blood for all conditions of infection and treatment. Infected untreated mice, infected antibiotic-treated mice and infected amoxicillin and dexamethasone-treated mice were compared. Treatment using both corticosteroids and antibiotics was associated with differential gene expression in the blood especially in Monocytes-Macrophages pathways. Depletion of these cells in infected mice was associated with a more severe bacterial infection and uncontrolled production of both pro-inflammatory and anti-inflammatory cytokines. Accordingly, children suffering from severe IMD had low counts of monocytes at admission. Our data are in favor of a role of corticosteroids in enhancing a polarization from pro-inflammatory to anti-inflammatory phenotypes of Monocytes-Macrophages axis that may help controlling meningococcal invasive infections.

The leading cause of hemolytic uremic syndrome (HUS) in children is Shiga toxin-producing Escherichia coli (STEC) infection, which has a major outbreak potential. Since the early 2010s, STEC epidemiology is characterized by a decline of the historically predominant O157 serogroup and the emergence of non-O157 STEC, especially O26 and O80 in France. STEC contamination occurs through the ingestion of contaminated food or water, person-to-person transmission, or contact with ruminants or their contaminated environment. The main symptom is diarrhea, which is bloody in about 60% of patients and occurs after a median incubation period of three days. Shiga toxins released by STEC induce a cascade of thrombogenic and inflammatory changes of microvascular endothelial cells. HUS is observed in 5-15% of STEC infection cases, defined by the triad of mechanical hemolytic anemia, thrombocytopenia, and acute renal injury. The diagnosis of STEC infection relies on biological screening for Shiga toxins and STEC in stools and serology. Treatment of STEC-HUS is mainly symptomatic, as no specific drug has proved effective. The effect of antibiotics in STEC infection and STEC-HUS remains debated; however, some bacteriostatic antibiotics might have a beneficial effect. Proofs of evidence of a benefit from complement blockade therapy in STEC-HUS are also lacking. Clinical and bacteriological STEC-HUS surveillance needs to be continued. Ongoing prospective studies will document the role of bacteriostatic antibiotics in STEC infection and STEC-HUS, and of complement blockade therapy in STEC-HUS.

Resume La documentation des infections bacteriennes chez le nouveau-ne represente un defi pour le clinicien et le microbiologiste a plusieurs titres. La difficulte de la realisation des prelevements chez le nouveau-ne augmente le risque de contamination des prelevements. Certains examens comme les hemocultures ont une sensibilite particulierement diminuee et ne peuvent etre reiteres en particulier chez le premature avant la mise sous antibiotique. Le prelevement des urines, simple chez le grand enfant, necessite un geste invasif chez le nouveau-ne qui n’est pas toujours realise. Par ailleurs les normes pour l’interpretation des examens ne sont pas aussi bien etablies que pour les autres patients. Tous ces elements, rendent souvent l’interpretation des examens difficile chez le nouveau-ne, avec pour consequence des antibiotherapies probabilistes poursuivies « dans le doute ». Des progres doivent etre imperativement realises sur les examens bacteriologiques en neonatologie en termes de faisabilite, de sensibilite, de rapidite et d’interpretation afin d’optimiser les prises en charge therapeutiques des nouveau-nes suspects d’infection bacterienne.

Shiga toxin-producing Escherichia coli (STEC) infection can cause simple diarrhoea but also life-threatening haemolytic and uraemic syndrome (HUS) in infants. Diagnosis is based on amplification of genes encoding the Shiga toxin ( stx ) on stools.1 The increasing ability to detect STEC due to the availability of multiplex PCR raises questions concerning the interpretation by clinicians of a positive PCR for patients with acute diarrhoea. Therefore, knowledge of the rate of asymptomatic carriage among young infants is needed. We assessed the rate of STEC carriage in a large cohort of children less than 2 years old, at risk of HUS in France, irrespective of their modality of care. This work is an ancillary study of a longitudinal survey of faecal Enterobacterales antimicrobial resistance performed in children aged 6–24 …

O80:H2 enterohemorrhagic Escherichia coli (EHEC) of sequence type ST301 is one of the main serotypes causing European hemolytic and uremic syndrome, but also invasive infections, due to extra-intestinal virulence factors (VFs). Here, we determined whether other such heteropathotypes exist among ST301. EnteroBase was screened for ST301 strains that were included in a general SNP-phylogeny. French strains belonging to a new heteropathotype clone were sequenced. ST, hierarchical clusters (HC), serotype, resistome, and virulome were determined using EnteroBase, the CGE website, and local BLAST. The ST301 general phylogeny shows two groups. Group A (n = 25) is mainly composed of enteropathogenic E. coli, whereas group B (n = 55) includes mostly EHEC. Three serotypes, O186:H2, O45:H2 and O55:H9, share the same virulome as one of the O80:H2 sub-clones from which they derive subsequent O-antigen switches. The O55:H9 clone, mainly present in France (n = 29), as well as in the UK (n = 5) and Germany (n = 1), has a low background of genetic diversity (four HC20), although it has three Stx subtypes, an H-antigen switch, and genes encoding the major extra-intestinal VF yersiniabactin, and extended-spectrum beta-lactamases. Diverse heteropathotype clones genetically close to the O80:H2 clone are present among the ST301, requiring close European monitoring, especially the virulent O55:H9 clone.

Objectives Mecillinam is recommended in France as a first-line treatment for lower urinary tract infections, due to the large increase in resistance of Escherichia coli to other oral treatments, such as co-trimoxazole or fluoroquinolones, its limited impact on faecal microbiota and its stability in the presence of numerous β-lactamases. However, we recently identified several mecillinam-resistant E. coli isolates with a high-level expression penicillinase (HEP) phenotype that merit further study. Patients and methods We studied two isogenic clinical isolates from one patient (one susceptible to mecillinam and one resistant to mecillinam) by WGS to determine the mechanism of mecillinam resistance and compared it with other mecillinam-resistant E. coli . We evaluated the synergistic combination of amoxicillin/clavulanate and mecillinam using a simple test, suitable for daily laboratory practice, to determine the MIC of this combination. Results We showed that the presence of an SNP in the promoter of the plasmidic TEM-1 β-lactamase gene is sufficient to confer resistance to mecillinam. This mechanism was present in 67% of HEP-phenotype E. coli tested. Combining mecillinam with amoxicillin/clavulanate abolished resistance, with an MIC compatible with clinical use. This association was not sensitive to the inoculum effect, in contrast to mecillinam alone. Conclusions An HEP phenotype can confer mecillinam resistance in vitro . This resistance is abolished, regardless of the inoculum, by combining mecillinam with amoxicillin/clavulanate, and can be easily tested in the laboratory. This combination may be used as an oral relay treatment of non-complicated pyelonephritis due to multiresistant E. coli strains.

Resume Les Escherichia.coli producteurs de Shiga-toxines ou E. coli enterohemorragiques (EHEC) sont responsables d’infections variees allant de la diarrhee aqueuse a la colite hemorragique pouvant evoluer vers un syndrome hemolytique et uremique chez l’enfant, principalement l’enfant de moins de 3 ans, ou une micro-angiopathie thrombotique chez l’adulte. La virulence de ces E. coli est associee a la presence de toxines appelees Shigatoxines. Les bovins constituent un important reservoir pour ces bacteries et l’homme se contamine par ingestion d’aliments contamines. Les EHEC sont consideres, comme des pathogenes emergents en sante publique, a l’origine de nombreuses epidemies de par le monde, consecutives principalement a la consommation d’aliments contamines. L’utilisation des antibiotiques est controversee pour le traitement de ces infections. La prevention de ces pathologies graves passe par des regles simples d’hygiene et de prevention alimentaire. Le diagnostic des EHEC repose sur l’isolement et la mise en evidence de leurs genes de virulence

We report an outbreak of Shiga toxin-producing Escherichia coli (STEC) associated paediatric haemolytic uraemic syndrome linked to the consumption of raw cow’s milk soft cheeses. From 25 March to 27 May 2019, 16 outbreak cases infected with STEC O26 (median age: 22 months) were identified. Interviews and trace-back investigations using loyalty cards identified the consumption of raw milk cheeses from a single producer. Trace-forward investigations revealed that these cheeses were internationally distributed.

We report an outbreak of Shiga toxin-producing Escherichia coli (STEC) associated paediatric haemolytic uraemic syndrome linked to the consumption of raw cow’s milk soft cheeses. From 25 March to 27 May 2019, 16 outbreak cases infected with STEC O26 (median age: 22 months) were identified. Interviews and trace-back investigations using loyalty cards identified the consumption of raw milk cheeses from a single producer. Trace-forward investigations revealed that these cheeses were internationally distributed.

Objectives Enterohaemorrhagic Escherichia coli (EHEC) infections may be complicated by haemolytic uraemic syndrome (HUS). The emerging worldwide EHEC serogroup O80 has acquired a mosaic plasmid combining extraintestinal virulence and antibiotic resistance. This hybrid pathotype is associated with invasive infections that require antibiotic therapy, classically not recommended in EHEC infections, increasing the risk of HUS. We characterized two ESBL-producing O80 EHEC strains, which is an unusual resistance mechanism among EHECs, and determined the safest therapy to be used for invasive infections. Methods WGS of two strains isolated from the stools of an asymptomatic carrier and a patient with HUS was performed using Illumina and Nanopore technologies. Generated reads were combined to assemble genomes. We determined the safest therapy by comparing Shiga toxin (Stx) production by the two strains in the presence of several antibiotics. Results The strains were genetically close to the O80 EHEC clone, belonging to ST301 and harbouring stx2d, eae-ξ, ehxA and genes characteristic of the extraintestinal virulence plasmid pS88. Long-read sequencing identified the acquisition of an additional plasmid harbouring CTX-M-type genes (blaCTX-M-14 and blaCTX-M-1). Azithromycin decreased Stx production at subinhibitory concentrations, ciprofloxacin increased it and imipenem had no major effect. The combination of azithromycin and imipenem overall reduced Stx production. Conclusions Acquisition of an additional plasmid harbouring ESBL genes is a step towards increasing the risk of O80 EHEC dissemination and represents a serious public health concern. The combination of azithromycin and imipenem reduced Stx production and suggests that this combination could be tested in clinical trials.

We developed a multiplex PCR method based on multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) that was designed for the rapid typing of Escherichia coli and Shigella isolates. The method amplifies seven VNTRs and does not require a sequencing capillary or fluorescent dyes. The amplification products are simply loaded on a standard agarose gel for electrophoresis, and the banding patterns are analyzed visually. We evaluated the method on 220 strains belonging to different collections: the E. coli reference (ECOR) collection ( n = 72), O1:K1 isolates causing neonatal meningitis ( n = 38), extended-spectrum beta-lactamase-producing fecal isolates belonging to the worldwide sequence type 131 (ST131) clone ( n = 38), Shiga toxin-producing E. coli (STEC) isolates of serogroups O157:H7 ( n = 21) and O26 ( n = 16, 8 of which belonged to an outbreak), 27 Shigella isolates (22 Shigella sonnei isolates, including 5 epidemic strains), and 8 reference strains. The performances were compared to those of multilocus sequence typing (MLST), the DiversiLab automated repetitive element palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). We found 66 different profiles among the isolates in the ECOR collection. Among the clonal group O1:K1 isolates, 14 different profiles were identified. For the 37 STEC isolates, we found 23 profiles, with 1 corresponding to the 8 epidemic strains. We found 19 profiles among the 27 Shigella isolates, with 1 corresponding to the epidemic strain. The method was able to recognize strains of the ST131 clone and to distinguish the O16 and O25b serogroups and identified 15 different MLVA types among them. This method allows the simple, fast, and inexpensive typing of E. coli/ Shigella isolates that can be carried out in any laboratory equipped for molecular biology and has a discriminatory power superior to that of MLST and DiversiLab REP-PCR but slightly lower than that of PFGE. IMPORTANCE Fast typing methods that can easily and accurately distinguish clonal groups and unrelated isolates are of particular interest for microbiologists confronted with outbreaks or performing epidemiological studies. Highly discriminatory universal methods, like PFGE, optical mapping, or WGS, are expensive and/or time-consuming. MLST is useful for phylogeny but is less discriminatory and requires sequencing facilities. PCR methods, which are fast and easy to perform, also have drawbacks. Random PCRs and REP-PCR are universal but lack reproducibility. Other PCR methods may lack the discriminatory power to differentiate isolates during outbreaks. MLVA combines the advantages of PCR methods with a high discriminatory power but in its standard form requires sequencing capillary electrophoresis. The method that we have developed combines the advantages of standard PCR (simple, fast, and inexpensive) with the high discriminatory power of MLVA and permits the typing of all E. coli isolates (either intestinal or extraintestinal pathogenic isolates as well as commensal isolates).

Epub 2019 Feb 26; International audience; Enterohaemorrhagic Escherichia coli (EHEC) are foodborne pathogens responsible for bloody diarrhoea and renal failure in humans. While Shiga toxin (Stx) is the cardinal virulence factor of EHEC, its production by E. coli is not sufficient to cause disease and many Shiga-toxin producing E. coli (STEC) strains have never been implicated in human infection. So far, the pathophysiology of EHEC infection is not fully understood and more knowledge is needed to characterize the "auxiliary" factors that enable a STEC strain to cause disease in humans. In this study, we applied a recombinase-based in vivo expression technology (RIVET) to the EHEC reference strain EDL933 in order to identify genes specifically induced during the infectious process, using mouse as an infection model. We identified 31 in vivo-induced (ivi) genes having functions related to metabolism, stress adaptive response and bacterial virulence or fitness. Eight of the 31 ivi genes were found to be heterogeneously distributed in EHEC strains circulating in France these last years. In addition, they are more prevalent in strains from the TOP 7 priority serotypes and particularly strains carrying significant virulence determinants such as Stx2 and intimin adhesin. This work sheds further light on bacterial determinants over-expressed in vivo during infection that may contribute to the potential of STEC strains to cause disease in humans.

Introduction Haemolytic uraemic syndrome (HUS) related to Shiga toxin-producing Escherichia coli (STEC) is the leading cause of acute renal failure in young children. In France, HUS surveillance in children aged Aim We present the results of this surveillance between 2007 and 2016. Methods A voluntary nationwide network of 32 paediatric departments notifies cases. Two national reference centres perform microbiological STEC confirmation. Results Over the study period, the paediatric HUS incidence rate (IR) was 1.0 per 100,000 children-years, with a median of 116 cases/year. In 2011, IR peaked at 1.3 per 100,000 children-years, and decreased to 1.0 per 100,000 children-years in 2016. STEC O157 associated HUS peaked at 37 cases in 2011 and decreased to seven cases in 2016. Cases of STEC O26-associated HUS have increased since 2010 and STEC O80 associated HUS has emerged since 2012, with 28 and 18 cases respectively reported in 2016. Four STEC-HUS food-borne outbreaks were detected (three STEC O157 linked to ground beef and raw-milk cheese and one STEC O104 linked to fenugreek sprouts). In addition, two outbreaks related to person-to-person transmission occurred in distinct kindergartens (STEC O111 and O26). Conclusions No major changes in HUS IRs were observed over the study period of 10 years. However, changes in the STEC serogroups over time and the outbreaks detected argue for continuing epidemiological and microbiological surveillance.

We developed a multiplex PCR method based on multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) that was designed for the rapid typing of Escherichia coli and Shigella isolates. The method amplifies seven VNTRs and does not require a sequencing capillary or fluorescent dyes. The amplification products are simply loaded on a standard agarose gel for electrophoresis, and the banding patterns are analyzed visually. We evaluated the method on 220 strains belonging to different collections: the E. coli reference (ECOR) collection (n = 72), O1:K1 isolates causing neonatal meningitis (n = 38), extended-spectrum beta-lactamase-producing fecal isolates belonging to the worldwide sequence type 131 (ST131) clone (n = 38), Shiga toxin-producing E. coli (STEC) isolates of serogroups O157:H7 (n = 21) and O26 (n = 16, 8 of which belonged to an outbreak), 27 Shigella isolates (22 Shigella sonnei isolates, including 5 epidemic strains), and 8 reference strains. The performances were compared to those of multilocus sequence typing (MLST), the DiversiLab automated repetitive element palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). We found 66 different profiles among the isolates in the ECOR collection. Among the clonal group O1:K1 isolates, 14 different profiles were identified. For the 37 STEC isolates, we found 23 profiles, with 1 corresponding to the 8 epidemic strains. We found 19 profiles among the 27 Shigella isolates, with 1 corresponding to the epidemic strain. The method was able to recognize strains of the ST131 clone and to distinguish the O16 and O25b serogroups and identified 15 different MLVA types among them. This method allows the simple, fast, and inexpensive typing of E. coli/Shigella isolates that can be carried out in any laboratory equipped for molecular biology and has a discriminatory power superior to that of MLST and DiversiLab REP-PCR but slightly lower than that of PFGE. IMPORTANCE Fast typing methods that can easily and accurately distinguish clonal groups and unrelated isolates are of particular interest for microbiologists confronted with outbreaks or performing epidemiological studies. Highly discriminatory universal methods, like PFGE, optical mapping, or WGS, are expensive and/or time-consuming. MLST is useful for phylogeny but is less discriminatory and requires sequencing facilities. PCR methods, which are fast and easy to perform, also have drawbacks. Random PCRs and REP-PCR are universal but lack reproducibility. Other PCR methods may lack the discriminatory power to differentiate isolates during outbreaks. MLVA combines the advantages of PCR methods with a high discriminatory power but in its standard form requires sequencing capillary electrophoresis. The method that we have developed combines the advantages of standard PCR (simple, fast, and inexpensive) with the high discriminatory power of MLVA and permits the typing of all E. coli isolates (either intestinal or extraintestinal pathogenic isolates as well as commensal isolates).

We report a hemolytic uremic syndrome (HUS) case due to Stx2f-producing E. coli illustrating the diagnostic difficulty of this Shiga-toxin subtype. Clinicians should be aware of limits of certain rapid molecular panels that are increasingly being used and may play a role in underestimating the global burden of such infections.

International audience; BACKGROUND: Prodromal symptoms are frequently reported in the atypical form of Hemolytic uremic syndrome (aHUS) suggesting implication of infectious triggers. Some pathogens may also play a role in the mechanisms of production of autoantibody directed against Factor H (FH), a complement regulator, leading to aHUS.METHODS: The presence of 15 gastrointestinal (GI) pathogens was investigated by using xTAG-based multiplex PCR techniques on stools collected at the acute phase in a cohort of Indian HUS children classified according to the presence or absence of anti-FH autoantibodies.RESULTS: Prevalence of pathogens in patients with anti-FH antibody (62.5%) was twice that in those without (31.5%). Different pathogens were detected, the most frequent being Clostridium difficile, Giardia intestinalis, Salmonella, Shigella, Rotavirus, Norovirus and Entamoeba histolytica. No stool was positive for Shigatoxin.CONCLUSION: This study reveals a higher prevalence of GI pathogens in anti-FH positive than in negative patients. No single pathogen was implicated exclusively in one form of HUS. These pathogens may play a role in the disease initiation by inducing complement activation or an autoimmune response.

Enterohemorrhagic Escherichia coli serogroup O80, involved in hemolytic uremic syndrome associated with extraintestinal infections, has emerged in France. We obtained circularized sequences of the O80 strain RDEx444, responsible for hemolytic uremic syndrome with bacteremia, and noncircularized sequences of 35 O80 E. coli isolated from humans and animals in Europe with or without Shiga toxin genes. RDEx444 harbored a mosaic plasmid, pR444_A, combining extraintestinal virulence determinants and a multidrug resistance– encoding island. All strains belonged to clonal complex 165, which is distantly related to other major enterohemorrhagic E. coli lineages. All stx-positive strains contained eae-ξ, ehxA, and genes characteristic of pR444_A. Among stx-negative strains, 1 produced extended-spectrum β-lactamase, 1 harbored the colistin-resistance gene mcr1, and 2 possessed genes characteristic of enteropathogenic and pyelonephritis E. coli. Because O80–clonal complex 165 strains can integrate intestinal and extraintestinal virulence factors in combination with diverse drug-resistance genes, they constitute dangerous and versatile multidrug-resistant pathogens We thank Roger Stephan and Lothar Beutin for providing the strains from Switzerland and Germany, respectively. This work was financed by Fonds d’Etudes et de Recherche du Corps Médical, AP-HP. Work in the Laboratorio de Referencia de Escherichia coli was financed by grant no. ED431C-2017-57 from Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia) and the European Regional Development Fund SI

Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that may be responsible for severe human infections. Only a limited number of serotypes, including O26:H11, are involved in the majority of serious cases and outbreaks. The main virulence factors, Shiga toxins (Stx), are encoded by bacteriophages. Seventy-four STEC O26:H11 strains of various origins (including human, dairy, and cattle) were characterized for their stx subtypes and Stx phage chromosomal insertion sites. The majority of food and cattle strains possessed the stx 1a subtype, while human strains carried mainly stx 1a or stx 2a . The wrbA and yehV genes were the main Stx phage insertion sites in STEC O26:H11, followed distantly by yecE and sbcB . Interestingly, the occurrence of Stx phages inserted in the yecE gene was low in dairy strains. In most of the 29 stx -negative E. coli O26:H11 strains also studied here, these bacterial insertion sites were vacant. Multilocus sequence typing of 20 stx -positive or stx -negative E. coli O26:H11 strains showed that they were distributed into two phylogenetic groups defined by sequence type 21 (ST21) and ST29. Finally, an EspK-carrying phage was found inserted in the ssrA gene in the majority of the STEC O26:H11 strains but in only a minority of the stx -negative E. coli O26:H11 strains. The differences in the stx subtypes and Stx phage insertion sites observed in STEC O26:H11 according to their origin might reflect that strains circulating in cattle and foods are clonally distinct from those isolated from human patients.

Extra-intestinal complications of Salmonella Typhi (S. Typhi) infections usually occur in endemic countries and in patients with underlying risk conditions. A 14-year-old immunocompetent girl was admitted with respiratory distress owing to S. Typhi pneumonia and pleural empyema. She was a native of Ivory Coast but had lived in France for 4 years and had not travelled abroad for several years. There were no gastro-intestinal symptoms and no S. Typhi carriage was detected in her family. She recovered completely with ceftriaxone and ciprofloxacin and pleural drainage was not required. An atypical presentation of S. Typhi should be considered even in settings where there are no risk factors.

Prodromal symptoms are frequently reported in the atypical form of Hemolytic uremic syndrome (aHUS) suggesting implication of infectious triggers. Some pathogens may also play a role in the mechanisms of production of autoantibody directed against Factor H (FH), a complement regulator, leading to aHUS.The presence of 15 gastrointestinal (GI) pathogens was investigated by using xTAG-based multiplex PCR techniques on stools collected at the acute phase in a cohort of Indian HUS children classified according to the presence or absence of anti-FH autoantibodies.Prevalence of pathogens in patients with anti-FH antibody (62.5%) was twice that in those without (31.5%). Different pathogens were detected, the most frequent being Clostridium difficile, Giardia intestinalis, Salmonella, Shigella, Rotavirus, Norovirus and Entamoeba histolytica. No stool was positive for Shigatoxin.This study reveals a higher prevalence of GI pathogens in anti-FH positive than in negative patients. No single pathogen was implicated exclusively in one form of HUS. These pathogens may play a role in the disease initiation by inducing complement activation or an autoimmune response.

Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR- assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains.

We describe a new atypical Shiga-toxin-producing Escherichia coli (STEC) responsible for a severe episode of haemolytic–uraemic syndrome in an adult with a relapse associated with bacteraemia. This STECs train of serotype O80:H2 harboured stx2c and stx2d gene subtypes, the rare eae ɛ variant and a ColV plasmid with a conserved virulence plasmidic region involved in virulence of human and avian extraintestinal pathogenic E. coli. This atypical hybrid pathotype, which represents a new threat, is a further demonstration that STEC may be a recipient for extraintestinal virulence factors and raises again the question of antibiotic therapy during STEC infection.

Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR-assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains.