Multicenter Preparedness Exercise Enables Rapid Development of Cluster-Specific PCR-Based Screening Assays from Bacterial Genomic Data.

PCR-based screening assays targeting strain-specific genetic markers allow the timely detection and specific differentiation of bacterial strains. Especially in situations where an infection cluster occurs, fast assay development is crucial for supporting targeted control measures. However, the turnaround times (TATs) for assay setup may be high due to insufficient knowledge about screening assay methods, workflows, and software tools. Here, two blind-coded and quality-controlled ring trials were performed in which five German laboratories established PCR-based screening assays from genomic data that specifically target selected bacterial clusters within two bacterial monospecies sample panels. While the first ring trial was conducted without a time limit to train the participants and assess assay feasibility, in the second ring trial, a challenging time limit of 2 weeks was set to force fast assay development as soon as genomic data were available. During both ring trials, we detected high interlaboratory variability regarding the screening assay methods and targets, the TATs for assay setup, and the number of screening assays. The participants designed between one and four assays per cluster that targeted cluster-specific unique genetic sequences, genes, or single nucleotide variants using conventional PCRs, high-resolution melting assays, or TaqMan PCRs. Assays were established within the 2-week time limit, with TATs ranging from 4 to 13 days. TaqMan probe delivery times strongly influenced TATs. In summary, we demonstrate that a specific exercise improved the preparedness to develop functional cluster-specific PCR-based screening assays from bacterial genomic data. Furthermore, the parallel development of several assays enhances assay availability.

[Assessment of available and currently applied typing methods of zoonotic pathogens using the example of Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC)]. / Bestandsaufnahme der verfügbaren und aktuell eingesetzten Typisierungsmethoden von zoonotischen Erregern am Beispiel von Shiga Toxin-bildenden bzw. enterohämorrhagischen Escherichia coli (STEC/EHEC).

INTRODUCTION: In order to improve patient care and to increase food safety within the framework of One Health, the project "Integrated Genomic Surveillance of Zoonotic Agents (IGS-Zoo)" aims to develop concepts for a genomic surveillance of Shiga toxin(Stx)-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany. METHODS: An online survey was conducted to assess the currently available and applied STEC/EHEC typing methods in the federal laboratories of veterinary regulation, food control, and public health service. RESULTS: Twenty-six questionnaires from 33 participants were evaluated with regard to STEC/EHEC. The number of STEC/EHEC-suspected samples that the laboratories process per year ranges between 10 and 3500, and out of these they obtain between 3 and 1000 pathogenic isolates. Currently the most frequently used typing method is the determination of Stx- and intimin-coding genes using polymerase chain reaction (PCR). Whole genome sequencing (WGS) is currently used by eight federal state laboratories, and nine are planning to implement it in the future. The most common obstacle for further typing of STEC/EHEC is that isolation from sample material is often unsuccessful despite apparent PCR detection of the stx genes. DISCUSSION: The results of the survey should facilitate the integration of the analysis methods developed in the project and emphasize the target groups' individual needs for corresponding training concepts.

Comparative phenotypic characterization of hybrid Shiga toxin-producing / uropathogenic Escherichia coli, canonical uropathogenic and Shiga toxin-producing Escherichia coli.

Hybrid Shiga toxin (Stx)-producing Escherichia coli (STEC) and uropathogenic E. coli (UPEC) strains are phylogenetically positioned between STEC and UPEC and can cause both diarrhea and urinary tract infections (UTIs). However, their virulence properties and adaptation to different host milieu in comparison to canonical UPEC and STEC strains are unknown. We determined phenotypes of the STEC/UPEC hybrid with respect to virulence including acid resistance, motility, biofilm formation, siderophore production, and adherence to human colonic Caco-2 and bladder T24 cells and compared to phenotypes of commensal strain MG1655, UPEC strain 536, and STEC strains B2F1 and Sakai. Moreover, we assessed the adaptation of the hybrid to artificial urine medium (AUM) and simulated colonic environment medium (SCEM). Overall acid resistance at pH 2.5 was high except in strains B2F1 and hybrid 05-00787 which showed reduced and extremely low acid resistance, respectively. Motility was reduced in hybrid 05-00787 and 09-05501 but strong in the remaining hybrids. While some hybrids showed high biofilm formation in LB, overall biofilm formation in SCEM and AUM were low and non-existent, respectively. All strains tested showed siderophore activity at equilibrium. All strains except MG1655 adhered to Caco-2 cells with the hybrid having similar adherence when compared to 536 but exhibited 2 and 3 times lower adherence when compared to B2F1 and Sakai, respectively. All Stx-producing strains adhered stronger to T24 cells than strains 536 and MG1655. Overall growth in LB, SCEM and AUM was consistent within the hybrid strains, except hybrid 05-00787 which showed significantly different growth patterns. Our data suggest that the hybrid is adapted to both, the intestinal and extraintestinal milieu. Expression of phenotypes typical of intestinal and extraintestinal pathogens thereby supports its potential to cause diarrhea and UTI.

Origin and Evolution of Hybrid Shiga Toxin-Producing and Uropathogenic Escherichia coli Strains of Sequence Type 141.

Hybrid Shiga toxin-producing Escherichia coli (STEC) and uropathogenic E. coli (UPEC) strains of multilocus sequence type 141 (ST141) cause both urinary tract infections and diarrhea in humans and are phylogenetically positioned between STEC and UPEC strains. We used comparative genomic analysis of 85 temporally and spatially diverse ST141 E. coli strains, including 14 STEC/UPEC hybrids, collected in Germany (n = 13) and the United States (n = 1) to reconstruct their molecular evolution. Whole-genome sequencing data showed that 89% of the ST141 E. coli strains either were STEC/UPEC hybrids or contained a mixture of virulence genes from other pathotypes. Core genome analysis and ancestral reconstruction revealed that the ST141 E. coli strains clustered into two lineages that evolved from a common ancestor in the mid-19th century. The STEC/UPEC hybrid emerged ∼100 years ago by acquiring an stx prophage, which integrated into previously unknown insertion site between rcsB and rcsD, followed by the insertion of a pathogenicity island (PAI) similar to PAI II of UPEC strain 536 (PAI II536-like). The two variants of PAI II536-like were associated with tRNA genes leuX and pheU, respectively. Finally, microevolution within PAI II536-like and acquisition of the enterohemorrhagic E. coli plasmid were observed. Our data suggest that intestinal pathogenic E. coli (IPEC)/extraintestinal pathogenic E. coli (ExPEC) hybrids are widespread and that selection pressure within the ST141 E. coli population led to the emergence of the STEC/UPEC hybrid as a clinically important subgroup. We hypothesize that ST141 E. coli strains serve as a melting pot for pathogroup conversion between IPEC and ExPEC, contrasting the classical theory of pathogen emergence from nonpathogens and corroborating our recent phenomenon of heteropathogenicity among pathogenic E. coli strains.

Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H- Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates.

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H- strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly, etp, and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H- strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins (stx2a and the cdtV-ABC operon) and adhesins (eae-γ, efa1, lpfAO157OI-141, and lpfAO157OI-154) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H- strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H- strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology.IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic-uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H- patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas.

Identification of Shiga Toxin-Producing Escherichia coli Outbreaks Using Whole Genome Sequencing.

Today, whole genome sequencing (WGS)-based typing is the gold standard approach to detect outbreaks of Shiga toxin-producing Escherichia coli (STEC) and to differentiate them from sporadic cases. Here, we describe an optimized protocol to efficiently determine the genome sequences of STEC using short read Illumina technology and provide information on helpful tools for the subsequent bioinformatic analysis.