Phylogenetic and Molecular Analysis of Food-Borne Shiga Toxin-Producing Escherichia coli

Seventy-five food-associated Shiga toxin-producing Escherichia coli (STEC) strains were analyzed by molecular and phylogenetic methods to describe their pathogenic potential. The presence of the locus of proteolysis activity (LPA), the chromosomal pathogenicity island (PAI) PAI ICL3, and the autotransporter- encoding gene sabA was examined by PCR. Furthermore, the occupation of the chromosomal integration sites of the locus of enterocyte effacement (LEE), selC, pheU, and pheV, as well as the Stx phage integration sites yehV, yecE, wrbA, z2577, and ssrA, was analyzed. Moreover, the antibiotic resistance phenotypes of all STEC strains were determined. Multilocus sequence typing (MLST) was performed, and sequence types (STs) and sequence type complexes (STCs) were compared with those of 42 hemolytic-uremic syndrome (HUS)-associated enterohemorrhagic E. coli (HUSEC) strains. Besides 59 STs and 4 STCs, three larger clusters were defined in this strain collection. Clusters A and C consist mostly of highly pathogenic eae-positive HUSEC strains and some related food-borne STEC strains. A member of a new O26 HUS-associated clone and the 2011 outbreak strain E. coli O104:H4 were found in cluster A. Cluster B comprises only eae-negative food-borne STEC strains as well as mainly eae-negative HUSEC strains. Although food-borne strains of cluster B were not clearly associated with disease, serotypes of important pathogens, such as O91:H21 and O113:H21, were in this cluster and closely related to the food-borne strains. Clonal analysis demonstrated eight closely related genetic groups of food-borne STEC and HUSEC strains that shared the same ST and were similar in their virulence gene composition. These groups should be considered with respect to their potential for human infection

Adding to Yersinia enterocolitica Gene Pool Diversity: Two Cryptic Plasmids from a Biotype 1A Isolate

We report the nucleotide sequence of two novel cryptic plasmids (4357 and 14 662 base pairs) carried by a Yersinia enterocolitica biotype 1A strain isolated from pork. As distinguished from most biotype 1A strains, this isolate, designated 07-04449, exhibited adherence to eukaryotic cells. The smaller plasmid pYe4449-1 carries five attributable open reading frames (ORFs) encoding the first CcdA/CcdB-like antitoxin/toxin system described for a Yersinia plasmid, a RepA-like replication initiation protein, and mobilizing factors MobA and MobC. The deduced amino acid sequences showed highest similarity to proteins described in Salmonella (CcdA/B), Klebsiella (RepA), and Plesiomonas (MobA/C) indicating genomic fluidity among members of the Enterobacteriaceae. One additional ORF with unknown function, termed ORF5, was identified with an ancestry distinct from the rest of the plasmid. While the C+G content of ORF5 is 38.3%, the rest of pYe4449-1 shows a C+G content of 55.7%. The C+G content of the larger plasmid pYe4449-2 (54.9%) was similar to that of pYe4449-1 (53.7%) and differed from that of the Y. enterocolitica genome (47.3%). Of the 14 ORFs identified on pYe4449-2, only six ORFs showed significant similarity to database entries. For three of these ORFs likely functions could be ascribed: a TnpR-like resolvase and a phage replication protein, localized each on a low C+G island, and DNA primase TraC. Two ORFs of pYe4449-2, ORF3 and ORF7, seem to encode secretable proteins. Epitope-tagging of ORF3 revealed protein expression at 4°C but not at or above 27°C suggesting adaptation to a habitat outside swine. The hypothetical protein encoded by ORF7 is the member of a novel repeat protein family sharing the DxxGN(x)nDxxGN motif. Our findings illustrate the exceptional gene pool diversity within the species Y. enterocolitica driven by horizontal gene transfer events

Shiga toxin producing Escherichia coli: identification of non-O157:H7-Super-Shedding cows and related risk factors

<p>Abstract</p> <p>Background</p> <p>Shiga toxin producing <it>Escherichia coli </it>(STEC) are an important cause of human gastro-enteritis and extraintestinal sequelae, with ruminants, especially cattle, as the major source of infection and reservoir. In this study, the fecal STEC shedding of 133 dairy cows was analyzed over a period of twelve months by monthly sampling with the aim to investigate shedding patterns and risk factors.</p> <p>Results</p> <p>Overall, 24.7% (in total 407) of 1,646 fecal samples were tested positive for <it>stx </it>by PCR with inner-herd prevalences on the different farms of 11.1% to 32.3%. At individual levels, cows were <it>stx</it>-positive on zero to eight consecutive samplings. According to a strictly longitudinal definition of Super-Shedding, in the present study 14 cows were identified as Super-Shedders of non-O157 serotypes.</p> <p>Significant risk factors for the shedding of STEC were the month of sampling, the number of lactations and days in lactation, the nutritional condition, the somatic cell count and the content of protein in milk. Most notably, the presence of STEC Super-Shedding cows in the herd was a significant risk factor, revealing that STEC Super-Shedding is not restricted to STEC O157:H7 alone.</p> <p>Conclusions</p> <p>These data have implications for possible interventions, as removing single non-O157:H7 STEC Super-Shedding cattle from farms would significantly reduce STEC burden.</p

COVID-19-Pandemie führte zu starkem Rückgang von darmpathogenen Erregern

Im Zuge der im März 2020 zur Bewältigung der COVID-19-Pandemie ergriffenen Infektionsschutzmaßnahmen wurde nicht nur ein abrupter Rückgang von Atemwegserkrankungen beobachtet, sondern im Verlauf des Jahres auch ein in diesem Maße nicht erwarteter Rückgang von bakteriellen Enteritiden. Abgebildet wurde dieser Trend durch die erfassten Fallzahlen im Meldesystem gemäß Infektionsschutzgesetz. Trotz einer geringeren Anzahl von Erkrankungsfällen bzw. Einsendungen konnte das Nationale Referenzzentrum für Salmonellen und andere bakterielle Enteritiserreger die integrierte molekulare Surveillance von ausgewählten lebensmittelbedingten darmpathogenen Infektionserregern (S. enterica, EHEC, L. monocytogenes, Shigella und Campylobacter) erfolgreich fortführen und ausbauen.Peer Reviewe

Enteropathisches hämolytisch-urämisches Syndrom: Sporadischer Einzelfall oder Teil eines Krankheitsausbruchs?

Das hämolytisch-urämische Syndrom (HUS) ist ein lebensbedrohlicher Zustand, charakterisiert durch akutes Nierenversagen, hämolytische Anämie und Thrombozytopenie. In >80% der Fälle liegen gastrointestinale Infektionen mit enterohämorrhagischen Escherichia coli (EHEC) zugrunde. Diese enterisch-infektiöse (enteropathische) Form des HUS tritt meist bei Kindern unter 6 Jahren auf. Da u. U. rasch eine Blutreinigung eingeleitet werden muss, sollten die Betroffenen in spezialisierten Kinderdialysezentren behandelt werden. Aufgrund der überwiegend enterisch-infektiösen Ätiologie können vereinzelt auftretende Fälle wichtige Hinweise auf Erkrankungshäufungen liefern. In Deutschland treten Infektionen mit einer seltenen Sorbitol fermentierenden Variante von EHEC O157 auf, die schon mehrfach in HUS-Krankheitsausbrüchen mit Todesfällen resultierten. Bereits der Verdacht sowie die Erkrankung oder der Tod an enteropathischem HUS sind durch den feststellenden Arzt unverzüglich an das zuständige Gesundheitsamt zu melden. Dadurch können zeitnah Maßnahmen zur Verhinderung der Ausbreitung der Infektion getroffen werden. Die epidemischen Aspekte des EHEC-assoziierten HUS werden dargestellt, und Kinderärzten Hilfestellungen im Hinblick auf eine frühzeitige Diagnose und Meldung gegeben.Hemolytic uremic syndrome (HUS) is a life-threatening condition characterized by acute renal failure, hemolytic anemia and thrombocytopenia. More than 80% of pediatric HUS is caused by infection with enterohemorrhagic Escherichia coli (EHEC). EHEC-associated HUS mainly affects children under six years of age. HUS patients should be treated in specialized clinics, which are able to provide dialysis. Importantly, sporadic cases of HUS can signalize outbreaks of EHEC infections. In Germany, a rare sorbitol-fermenting variant of EHEC O157 caused several large HUS outbreaks with fatal cases. Physicians are required to notify every suspected, confirmed or fatal case of HUS to their local health department, thereby enabling public health authorities to intervene promptly. This article describes the epidemic aspects of EHEC-associated HUS and assists physicians and pediatricians in terms of timely diagnosis and notification

Correlation between the genomic o454-nlpD region polymorphisms, virulence gene equipment and phylogenetic group of extraintestinal Escherichia coli (ExPEC) enables pathotyping irrespective of host, disease and source of isolation

Background: The mutS-rpoS intergenic region in E. coli displays a mosaic structure which revealed pathotype specific patterns. To assess the importance of this region as a surrogate marker for the identification of highly virulent extraintestinal pathogenic E. coli (ExPEC) strains we aimed to: (i) characterize the genetic diversity of the mutS gene and the o454-nlpD genomic region among 510 E. coli strains from animals and humans; (ii) delineate associations between the polymorphism of this region and features such as phylogenetic background of E. coli, pathotype, host species, clinical condition, serogroup and virulence associated genes (VAG)s; and (iii) identify the most important VAGs for classification of the o454-nlpD region. Methods: Size variation in the o454-nlpD region was investigated by PCR amplification and sequencing. Phylogenetic relationships were assessed by Ecor- and Multilocus sequence- typing (MLST), and a comparative analysis between mutS gene phylogenetic tree obtained with RAxML and the MLST grouping method was performed. Correlation between o454-nlpD patterns and the features described above were analysed. In addition, the importance of 47 PCR-amplified ExPEC-related VAGs for classification of o454-nlpD patterns was investigated by means of Random Forest algorithm. Results: Four main structures (patterns I-IV) of the o454-nlpD region among ExPEC and commensal E. coli strains were identified. Statistical analysis showed a positive and exclusive association between pattern III and the ExPEC strains. A strong association between pattern III and either the Ecor group B2 or the sequence type complexes known to represent the phylogenetic background of highly virulent ExPEC strains (such as STC95, STC73 and STC131) was found as well. RF analyses determined five genes (csgA, malX, chuA, sit, and vat) to be suitable to predict pattern III strains. Conclusion: The significant association between pattern III and group B2 strains suggested the o454-nlpD region to be of great value in identifying highly virulent strains among the mixed population of E. coli promising to be the basis of a future typing tool for ExPEC and their gut reservoir. Furthermore, top-ranked VAGs for classification and prediction of pattern III were identified. These data are most valuable for defining ExPEC pathotype in future in vivo assays

Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli

Multilocus sequence typing of 169 non-O157 enterohemorrhagic Escherichia coli (EHEC) isolated from patients with hemolytic uremic syndrome (HUS) demonstrated 29 different sequence types (STs); 78.1% of these strains clustered in 5 STs. From all STs and serotypes identified, we established a reference panel of EHEC associated with HUS (HUSEC collection).</p

Diversity of Salmonella enterica serovar Derby isolated from pig, pork and humans in Germany

Salmonella enterica serovar Derby (S. Derby) is one of the most prevalent serovars in pigs in Europe and in the U.S. and ranks among the 10 most frequently isolated serovars in humans. Therefore, a set of 82 epidemiologically unrelated S. Derby strains isolated between 2006 and 2008 from pigs, pork and humans in Germany was selected and investigated in respect to the transmission of clonal groups of the serovar along the food chain. Various phenotypic and genotypic methods were applied and the pathogenicity and resistance gene repertoire was determined. Phenotypically 72% of the strains were susceptible to all 17 antimicrobials tested while the others were monoresistant to tetracycline or multi-resistant with different resistance profiles. Four major clonal groups were identified based on PFGE, sequence data of the virulence genes sopA, sopB and sopD, VNTR-locus STTR5 and MLST revealing also the new sequence type ST774. Thirty different PFGE profiles were detected resulting in four clusters representing the four groups. The pathogenicity gene repertoire of 32 representative S. Derby strains analyzed by microarray showed six types with differences in the Salmonella pathogenicity islands, pathogenicity genes on smaller islets or prophages and fimbriae coding genes. The pathogenicity gene repertoire of the predominant types PAT DE1 and DE2 were most similar to the ones of S. Paratyphi B (dT+, O5−) and to a minor degree to S. Infantis and S. Virchow PATs. Overall this study showed that in Germany currently one major S. Derby clone is frequently isolated from pigs and humans. Contaminated pork was identified as one vehicle and consequently is a risk for human health. To prevent this serovar from entering the food chain, control measurements should be applied at the farm level

Genetic diversity and pathogenic potential of Shiga toxin-producing Escherichia coli (STEC) derived from German flour

Shiga toxin-producing Escherichia coli (STEC) can cause severe human illness, which are frequently linked to the consumption of contaminated beef or dairy products. However, recent outbreaks associated with contaminated flour and undercooked dough in the United States and Canada, highlight the potential of plant based food as transmission routes for STEC. In Germany STEC has been isolated from flour, but no cases of illness have been linked to flour. In this study, we characterized 123 STEC strains isolated from flour and flour products collected between 2015 and 2019 across Germany. In addition to determination of serotype and Shiga toxin subtype, whole genome sequencing (WGS) was used for isolates collected in 2018 to determine phylogenetic relationships, sequence type (ST), and virulence-associated genes (VAGs). We found a high diversity of serotypes including those frequently associated with human illness and outbreaks, such as O157:H7 (stx2c/d, eae), O145:H28 (stx2a, eae), O146:H28 (stx2b), and O103:H2 (stx1a, eae). Serotypes O187:H28 (ST200, stx2g) and O154:H31 (ST1892, stx1d) were most prevalent, but are rarely linked to human cases. However, WGS analysis revealed that these strains, as well as, O156:H25 (ST300, stx1a) harbour high numbers of VAGs, including eae, nleB and est1a/sta1. Although STEC-contaminated flour products have yet not been epidemiologically linked to human clinical cases in Germany, this study revealed that flour can serve as a vector for STEC strains with a high pathogenic potential. Further investigation is needed to determine the sources of STEC contamination in flour and flour products particularly in regards to these rare serotypes.Peer Reviewe