Genetic characterization of Shigatoxigenic and enteropathogenic Escherichia coli O80:H2 from diarrhoeic and septicaemic calves and relatedness to human Shigatoxigenic E. coli O80:H2.

AIM: The purpose of this work was to identify and genetically characterize enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) O80:H2 from diarrhoeic and septicaemic calves in Belgium and to comparing them with human EHEC after whole genome sequencing. METHODS AND RESULTS: Ten EHEC and 21 EPEC O80 identified by PCR between 2009 and 2018 from faeces, intestinal content and a kidney of diarrhoeic or septicaemic calves were genome sequenced and compared to 19 human EHEC identified between 2008 and 2019. They all belonged to the O80:H2 serotype and ST301, harboured the eaeξ gene, and 23 of the 29 EHEC contained the stx2d gene. Phylogenetically, they were distributed in two major sub-lineages: one comprised a majority of bovine EPEC whereas the second one comprised a majority of stx2d bovine and human EHEC. CONCLUSIONS: Not only EPEC but also EHEC O80:H2 are present in diarrhoeic and septicaemic calves in Belgium and are genetically related to human EHEC. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings support the need to assess cattle as potential source of contamination of humans by EHEC O80:H2 and to understand the evolution of bovine and human EHEC and EPEC O80:H2.

Characteristics of Shiga toxin producing- and enteropathogenic Escherichia coli of the emerging serotype O80:H2 isolated from humans and diarrhoeic calves in Belgium.

OBJECTIVES: Recently a highly virulent Escherichia coli O80:H2 pathotype carrying Shiga toxin genes, the intimin subtype eaeξ, and genes associated with the extraintestinal pathogenic E. coli (ExPEC) pS88 plasmid was described in France. In this study we examine the relatedness of Belgian E. coli O80:H2 isolated from humans and diarrhoeic calves as well their similarities with the French pathotype. METHODS: Eighteen Belgian E. coli O80:H2 strains (nine human Shiga toxin-producing E. coli (STEC) (2008-2016), two bovine STEC (1987) and seven bovine atypical enteropathogenic E. coli (aEPEC) (2009-2015)) were characterized with conventional PCR, disc diffusion susceptibility testing and whole genome sequencing. RESULTS: Only nine sporadic human STEC O80:H2 cases have been detected in Belgium. All patients were female, just two of them suffered from haemolytic uremic syndrome. All studied strains had the eaeξ subtype, belonged to the multi-locus sequence type ST-301, and carried virulence genes associated with the type III secretion system and effectors not encoded by the locus of enterocyte effacement (LEE). Multiple genes of the pS88 plasmid were detected in all but two strains (one human and one calf STEC). The Shiga toxin subtypes stx1a (n = 3; one human, two calf), stx2a (n = 2) and stx2d (n = 6) were detected. All strains were multidrug resistant, two were extended-spectrum ß-lactamase positive. Core genome MLST revealed that some human and calf E. coli differed by only 22 loci. CONCLUSIONS: The STEC/ExPEC O80:H2 pathotype was present in calves in Belgium as early as 1987, but human infections have been rare and mostly mild. The human STEC and bovine aEPEC cluster together and have the potential to be as virulent as the French isolates, as shown by their similar gene content.

Risk determinants for the development of typical haemolytic uremic syndrome in Belgium and proposition of a new virulence typing algorithm for Shiga toxin-producing Escherichia coli.

In Belgium, it is mandatory to report Shiga toxin-producing Escherichia coli (STEC) infections to the health inspection authorities. To facilitate the decision making regarding infection control measures, information about the risk factors for the development of the haemolytic uremic syndrome (HUS) can be helpful. We performed statistical analyses on a dataset of 411 Belgian STEC strains. Demographic and clinical patient characteristics as well as phenotypical and genotypical STEC strain characteristics were taken into account. Multivariate logistic regression models indicated that age categories ⩽5, 6-12 and ⩾75; the stx2 gene; and the eae gene were significant HUS development risk determinants. The stx2a subtype had the highest risk (OR 29.6, 95% CI 7.0-125.1), while all stx1 subtypes encompassed a significant lower risk (OR 0.3, 95% CI 0.1-0.5). Presence of the stx1 gene without stx2 encompassed a lower risk than the combined presence of stx1 and stx2, or stx2 solely. Based on these results, we propose a new virulence typing algorithm that will enable the National Reference Centre to provide the physicians and health inspection authorities with a risk classification for the development of HUS. We believe this will contribute to a more efficient STEC infection control management in Belgium.

Haemolytic uremic syndrome surveillance in children less than 15 years in Belgium, 2009-2015.

BACKGROUND: The Haemolytic Uremic Syndrome (HUS) is the most severe manifestation of infection with Shiga toxin-producing Escherichia coli (STEC). In Belgium, the surveillance of paediatric HUS cases is conducted by a sentinel surveillance network of paediatricians called Pedisurv. In this article, we present the main findings of this surveillance from 2009 to 2015 and we describe an annual incidence of HUS. METHODS: For each case of HUS <  15 years notified by the paediatricians, clinical, microbiological and epidemiological data were collected by a questionnaire. National hospital discharge data with ICD-9 code 283.11 were used to calculate the incidence of HUS in children < 15 years. RESULTS: From 2009 to 2015, 110 cases were notified to the Pedisurv network with a mean annual notification rate of 0.8/100,000 in children < 15 years. Death occurred in 2.5% of all patients and the median number of days of hospitalization was 10 days. One third (35.4%) of the HUS cases were confirmed positive STEC, with a majority of STEC O157. The mean annual incidence based on the hospital discharge data was 3.2/100,000 in children < 15 years and 4.5/100,000 in children < 5 years. CONCLUSION: The incidence of paediatric HUS in Belgium is high compared to other European countries. Its surveillance in Belgium is quite comprehensive and, although less effective than monitoring all STEC infections to detect the emergence of outbreaks, is important to better monitor circulation of the most pathogenic STEC strains. In this context, efforts are still needed to send samples and STEC strains from HUS cases to the National Reference Centre.

Low prevalence of the 'gang of seven' and absence of the O80:H2 serotypes among Shigatoxigenic and enteropathogenic Escherichia coli (STEC and EPEC) in intestinal contents of healthy cattle at two slaughterhouses in Belgium in 2014.

AIMS: The purpose of this survey was to estimate the respective prevalence of the 'gang of seven' and 'non-gang of seven' serotypes of Shigatoxigenic and enteropathogenic Escherichia coli and to identify the O80:H2 serotype in 245 intestinal contents collected at two slaughterhouses in Belgium in 2014. METHODS AND RESULTS: After overnight enrichment growth, the 69 intestinal contents testing positive with PCR targeting the eae, stx1 and stx2 genes were inoculated onto four agar media. Of the 2542 colonies picked up, 677 from 59 samples were PCR confirmed. The most frequent virulotypes were eae+ in 47 (80%) samples, stx2+ in 20 (34%) samples and eae+ stx1+ in 16 (27%) samples. PCR-positive colonies belonged to different virulotypes in 36 samples. No colony was O80-positive, whereas two eae+ colonies from two samples were O26:H11, 50 eae+ stx1+ and eae+ from eight samples were O103:H2 and two eae+ stx1+ stx2+ colonies from one sample were O157:H7. CONCLUSIONS: The 'non-gang of seven' serotypes are more frequent than the 'gang of seven' serotypes and the O80:H2 serotype was not detected among Shigatoxigenic and enteropathogenic Escherichia coli in the intestines of cattle at these two slaughterhouses. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the identification protocols of Shigatoxigenic Escherichia coli focus on the 'gang of seven' serotypes, several other serotypes can be present with possible importance in public health. Innovative selective identification procedures should be designed.

Detection of Shiga toxin-producing and other diarrheagenic Escherichia coli by the BioFire FilmArray® Gastrointestinal Panel in human fecal samples.

The purpose of this investigation was the evaluation of the performance of the BioFire FilmArray® Gastrointestinal (FA-GI) Panel, a multiplexed molecular stool screening assay, for the detection of diarrheagenic Escherichia coli (DEC), with emphasis on Shiga toxin-producing E. coli (STEC). A dilution series of 12 STEC reference strains was tested with the FA-GI Panel to assess the analytical sensitivity. A total of 389 patient samples were analyzed with the FA-GI Panel and confirmation of the detected DEC was attempted with an in-house culture-based polymerase chain reaction (PCR) method. All Shiga toxin genes, except the one encoding Stx2f, were detected in bacterial dilutions ranging from 10(4) to 10(2) colony-forming units (CFU)/ml. eae + stx2f + STEC was misclassified as enteropathogenic E. coli (EPEC). Different sensitivities for various gene targets present in one isolate led to differing identifications depending on the concentration. Using the in-house method as a reference, the FA-GI Panel had a sensitivity of 90.6 % [confidence interval (CI) 75.0 %-98.0 %] and a specificity of 97.2 % (CI 94.9 %-98.6 %) for STEC detection in feces. At least one DEC was reported in 35.5 % (171/389) of the patient specimens, with EPEC being the most prevalent (n = 71). Only 59.7 % of the detected DEC could be confirmed, presumably because the comparator method was not applied directly on feces. The FA-GI Panel could not detect the stx2f subtype, misclassified certain pathogens, and the high detection rate of EPEC needs further investigation. Nevertheless, we believe that this sensitive and convenient system will prove to be an invaluable tool for the rapid diagnosis of most DEC infections, but culturing of the detected microorganisms should always be attempted.

Enteroaggregative Shiga toxin-producing Escherichia coli of serotype O104:H4 in Belgium and Luxembourg.

In 2011, a large outbreak of infections caused by Shiga toxin-producing Escherichia coli (STEC) O104:H4 occurred in Germany. This exceptionally virulent strain combined virulence factors of enteroaggregative E. coli (EAggEC) and STEC. After the outbreak only a few sporadic cases of infection with this rare serotype were reported, most of which were related to travel to the Middle East or North Africa. Here we describe two cases of enteroaggregative STEC (Agg-STEC) O104:H4 infection that occurred in Belgium in 2012 and 2013 respectively. In both cases travel in a Mediterranean country preceded the infection. The first strain was isolated from the stool of a 42-year-old woman presenting bloody diarrhoea, who had travelled to Tunisia the week before. The second case involves a 14-year-old girl who, upon her return from Turkey to Belgium, suffered from an episode of bloody diarrhoea and haemolytic uraemic syndrome. Extended typing of the isolates with pulsed field gel electrophoresis revealed that the strains were closely related, though not exactly the same as the 2011 outbreak strain. This report supports the previously made hypothesis that Agg-STEC has a human reservoir and might be imported by travellers coming from an area where the pathogen is endemic. Furthermore, it emphasizes the concern that these bacteria may cause future outbreaks as evenly virulent O104:H4 isolates seem to be widespread.