Salmonella enterica is the second most reported bacterial cause of food-borne infections in Europe. Therefore molecular surveillance activities based on pathogen subtyping are an important measure of ...controlling Salmonellosis by public health agencies. In Germany, at the federal level, this work is carried out by the National Reference Center for Salmonella and other Bacterial Enteric Pathogens (NRC). With rise of next generation sequencing techniques, the NRC has introduced whole-genome-based typing methods for S. enterica in 2016. In this study we report on the feasibility of genome-based in silico serotyping in the German setting using raw sequence reads. We found that SeqSero and seven gene MLST showed 98% and 95% concordance, respectively, with classical serotyping for the here evaluated serotypes, including the most common German serotypes S. Enteritidis and S. Typhimurium as well as less frequently found serotypes. The level of concordance increased to >99% when the results of both in silico methods were combined. However, both tools exhibited misidentification of monophasic variants, in particular monophasic S. Typhimurium and therefore need to be fine-tuned for reliable detection of this epidemiologically important variant. We conclude that with adjustments Salmonella genome-based serotyping might become the new gold standard.
Shiga toxin-producing
(STEC) and the STEC subgroup enterohemorrhagic
cause intestinal infections with symptoms ranging from watery diarrhea to hemolytic-uremic syndrome (HUS). A key tool for the ...epidemiological differentiation of STEC is serotyping. The serotype in combination with the main virulence determinants gives important insight into the virulence potential of a strain. However, a large fraction of STEC strains found in human disease, including strains causing HUS, belongs to less frequently detected STEC serovars or their O/H antigens are unknown or even untypeable. Recent implementation of whole-genome sequence (WGS) analysis, in principle, allows the deduction of serovar and virulence gene information. Therefore, here we compared classical serovar and PCR-based virulence marker detection with WGS-based methods for 232 STEC strains, focusing on less frequently detected STEC serovars and nontypeable strains. We found that the results of WGS-based extraction showed a very high degree of overlap with those of the more classical methods. Specifically, the rate of concordance was 97% for O antigens (OAGs) and 99% for H antigens (HAGs) of typeable strains and >99% for
,
, or
for all strains. Ninety-eight percent of nontypeable OAGs and 100% of nontypeable HAGs were defined by WGS analysis. In addition, the novel methods enabled a more complete analysis of strains causing severe clinical symptoms and the description of four novel STEC OAG loci. In conclusion, WGS is a promising tool for gaining serovar and virulence gene information, especially from a public health perspective.
Summary Background In an ongoing outbreak of haemolytic uraemic syndrome and bloody diarrhoea caused by a virulent Escherichia coli strain O104:H4 in Germany (with some cases elsewhere in Europe and ...North America), 810 cases of the syndrome and 39 deaths have occurred since the beginning of May, 2011. We analysed virulence profiles and relevant phenotypes of outbreak isolates recovered in our laboratory. Methods We analysed stool samples from 80 patients that had been submitted to the National Consulting Laboratory for Haemolytic Uraemic Syndrome in Münster, Germany, between May 23 and June 2, 2011. Isolates were screened with standard PCR for virulence genes of Shiga-toxin-producing E coli and a newly developed multiplex PCR for characteristic features of the outbreak strain ( rfbO104 , fliCH4 , stx2 , and terD ). Virulence profiles of the isolates were determined with PCR targeting typical virulence genes of Shiga-toxin-producing E coli and of other intestinal pathogenic E coli . We sequenced stx with Sanger sequencing and measured Shiga-toxin production, adherence to epithelial cells, and determined phylogeny and antimicrobial susceptibility. Findings All isolates were of the HUSEC041 clone (sequence type 678). All shared virulence profiles combining typical Shiga-toxin-producing E coli ( stx2 , iha, lpfO26 , lpfO113 ) and enteroaggregative E coli ( aggA, aggR, set1, pic, aap ) loci and expressed phenotypes that define Shiga-toxin-producing E coli and enteroaggregative E coli , including production of Shiga toxing 2 and aggregative adherence to epithelial cells. Isolates additionally displayed an extended-spectrum β-lactamase phenotype absent in HUSEC041. Interpretation Augmented adherence of the strain to intestinal epithelium might facilitate systemic absorption of Shiga toxin and could explain the high progression to haemolytic uraemic syndrome. This outbreak demonstrates that blended virulence profiles in enteric pathogens, introduced into susceptible populations, can have extreme consequences for infected people. Funding German Federal Ministry of Education and Research, Network Zoonoses.
We describe an outbreak of gastroenteritis and the hemolytic-uremic syndrome caused by Shiga-toxin-producing Escherichia coli in Germany in May, June, and July, 2011. The consumption of sprouts was ...identified as the most likely vehicle of infection.
We analyzed data from reports in Germany of Shiga-toxin-producing E. coli gastroenteritis and the hemolytic-uremic syndrome and clinical information on patients presenting to Hamburg University Medical Center (HUMC). An outbreak case was defined as a reported case of the hemolytic-uremic syndrome or of gastroenteritis in a patient infected by Shiga-toxin-producing E. coli, serogroup O104 or serogroup unknown, with an onset of disease during the period from May 1 through July 4, 2011, in Germany.
A total of 3816 cases (including 54 deaths) were reported in Germany, 845 of which (22%) involved the hemolytic-uremic syndrome. The outbreak was centered in northern Germany and peaked around May 21 to 22. Most of the patients in whom the hemolytic-uremic syndrome developed were adults (88%; median age, 42 years), and women were overrepresented (68%). The estimated median incubation period was 8 days, with a median of 5 days from the onset of diarrhea to the development of the hemolytic-uremic syndrome. Among 59 patients prospectively followed at HUMC, the hemolytic-uremic syndrome developed in 12 (20%), with no significant differences according to sex or reported initial symptoms and signs. The outbreak strain was typed as an enteroaggregative Shiga-toxin-producing E. coli O104:H4, producing extended-spectrum beta-lactamase.
In this outbreak, caused by an unusual E. coli strain, cases of the hemolytic-uremic syndrome occurred predominantly in adults, with a preponderance of cases occurring in women. The hemolytic-uremic syndrome developed in more than 20% of the identified cases.
Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause ...of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors, we provide new insights into the pathogenesis of EHEC O157 infections. Our data have implications for considering O157 OMVs as vaccine candidates.
Shiga toxin-producing E. coli (STEC), including the subgroup of enterohemorrhagic E. coli (EHEC), are important bacterial pathogens which cause diarrhea and the severe clinical manifestation ...hemolytic uremic syndrome (HUS). Genomic surveillance of STEC/EHEC is a state-of-the-art tool to identify infection clusters and to extract markers of circulating clinical strains, such as their virulence and resistance profile for risk assessment and implementation of infection prevention measures. The aim of the study was characterization of the clinical STEC population in Germany for establishment of a reference data set. To that end, from 2020 to 2022 1257 STEC isolates, including 39 of known HUS association, were analyzed and lead to a classification of 30.4 % into 129 infection clusters. Major serogroups in all clinical STEC analyzed were O26, O146, O91, O157, O103, and O145; and in HUS-associated strains were O26, O145, O157, O111, and O80. stx1 was less frequently and stx2 or a combination of stx, eaeA and ehxA were more frequently found in HUS-associated strains. Predominant stx gene subtypes in all STEC strains were stx1a (24 %) and stx2a (21 %) and in HUS-associated strains were mainly stx2a (69 %) and the combination of stx1a and stx2a (12.8 %). Furthermore, two novel O-antigen gene clusters (RKI6 and RKI7) and strains of serovars O45:H2 and O80:H2 showing multidrug resistance were detected. In conclusion, the implemented surveillance tools now allow to comprehensively define the population of clinical STEC strains including those associated with the severe disease manifestation HUS reaching a new surveillance level in Germany.
The zoonotic pathogen Campylobacter jejuni is among the leading causes of foodborne diseases worldwide. While C. jejuni colonises many wild animals and livestock, persistence mechanisms enabling the ...bacterium to adapt to host species' guts are not fully understood. In order to identify putative determinants influencing host preferences of distinct lineages, bootstrapping based on stratified random sampling combined with a k-mer-based genome-wide association was conducted on 490 genomes from diverse origins in Germany and Canada. We show a strong association of both the core and the accessory genome characteristics with distinct host animal species, indicating multiple adaptive trajectories defining the evolution of C. jejuni lifestyle preferences in different ecosystems. Here, we demonstrate that adaptation towards a specific host niche ecology is most likely a long evolutionary and multifactorial process, expressed by gene absence or presence and allele variations of core genes. Several host-specific allelic variants from different phylogenetic backgrounds, including dnaE, rpoB, ftsX or pycB play important roles for genome maintenance and metabolic pathways. Thus, variants of genes important for C. jejuni to cope with specific ecological niches or hosts may be useful markers for both surveillance and future pathogen intervention strategies.
Salmonella enterica subsp. enterica serovar 4,5,12:i:- is a monophasic variant of S. enterica serovar Typhimurium (antigenic formula 4,5,12:i:1,2). Worldwide, especially in several European countries ...and the United States, it has been reported among the 10 most frequently isolated serovars in pigs and humans. In the study reported here, 148 strains of the monophasic serovar isolated from pigs, pork, and humans in 2006 and 2007 in Germany were characterized by various phenotypic and genotypic methods. This characterization was done in order to investigate their clonality, the prevalence of identical subtypes in pigs, pork, and humans, and the genetic relatedness to other S. enterica serovar Typhimurium subtypes in respect to the pathogenic and resistance gene repertoire. Two major clonal lineages of the monophasic serovar were detected which can be differentiated by their phage types and pulsed-field gel electrophoresis (PFGE) profiles. Seventy percent of the strains tested belonged to definite phage type DT193, and those strains were mainly assigned to PFGE cluster B. Nineteen percent of the strains were typed to phage type DT120 and of these 86% belonged to PFGE cluster A. Sixty-five percent of the isolates of both lineages carried core multiresistance to ampicillin, streptomycin, tetracycline, and sulfamethoxazole encoded by the genes blaTEM₁₋like, strA-strB, tet(B), and sul2. No correlation to the source of isolation was observed in either lineage. Microarray analysis of 61 S. enterica serovar 4,5,12:i:- and 20 S. enterica serovar Typhimurium isolates tested determining the presence or absence of 102 representative pathogenicity genes in Salmonella revealed no differences except minor variations in single strains within and between the serovars, e.g., by presence of the virulence plasmid in four strains. Overall the study indicates that in Germany S. enterica serovar 4,5,12:i:- strains isolated from pig, pork, and human are highly related, showing their transmission along the food chain. Since the pathogenicity gene repertoire is highly similar to that of S. enterica serovar Typhimurium, it is essential that interventions are introduced at the farm level in order to limit human infection.
The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and haemolytic uraemic syndrome secretes blended virulence factors of enterohaemorrhagic and ...enteroaggregative E. coli, but their secretion pathways are unknown. We demonstrate that the outbreak strain releases a cocktail of virulence factors via outer membrane vesicles (OMVs) shed during growth. The OMVs contain Shiga toxin (Stx) 2a, the major virulence factor of the strain, Shigella enterotoxin 1, H4 flagellin, and O104 lipopolysaccharide. The OMVs bind to and are internalised by human intestinal epithelial cells via dynamin-dependent and Stx2a-independent endocytosis, deliver the OMV-associated virulence factors intracellularly and induce caspase-9-mediated apoptosis and interleukin-8 secretion. Stx2a is the key OMV component responsible for the cytotoxicity, whereas flagellin and lipopolysaccharide are the major interleukin-8 inducers. The OMVs represent novel ways for the E. coli O104:H4 outbreak strain to deliver pathogenic cargoes and injure host cells.
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