Eva Møller Nielsen

Real-Time Whole-Genome Sequencing for Routine Typing, Surveillance, and Outbreak Detection of Verotoxigenic Escherichia coli

ABSTRACT Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-producing Escherichia coli (VTEC). In Denmark, the Statens Serum Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week period in the fall of 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, multilocus sequence type (MLST) typing, and determination of phylogenetic relationship, and a specific VirulenceFinder for detection of E. coli virulence genes was developed as part of this study. In total, 46 suspected VTEC isolates were characterized in parallel during the study. VirulenceFinder proved successful in detecting virulence genes included in routine typing, explicitly verocytotoxin 1 (vtx1), verocytotoxin 2 (vtx2), and intimin (eae), and also detected additional virulence genes. VirulenceFinder is also a robust method for assigning verocytotoxin (vtx) subtypes. A real-time clustering of isolates in agreement with the epidemiology was established from WGS, enabling discrimination between sporadic and outbreak isolates. Overall, WGS typing produced results faster and at a lower cost than the current routine. Therefore, WGS typing is a superior alternative to conventional typing strategies. This approach may also be applied to typing and surveillance of other pathogens.

Fresh Chicken as Main Risk Factor for Campylobacteriosis, Denmark

Increased consumption of fresh poultry in Denmark has contributed substantially to the increasing incidence of human campylobacteriosis.

International Spread of an Epidemic Population of Salmonella enterica Serotype Kentucky ST198 Resistant to Ciprofloxacin

National Salmonella surveillance systems from France, England and Wales, Denmark, and the United States identified the recent emergence of multidrug-resistant isolates of Salmonella enterica serotype Kentucky displaying high-level resistance to ciprofloxacin. A total of 489 human cases were identified during the period from 2002 (3 cases) to 2008 (174 cases). These isolates belonged to a single clone defined by the multilocus sequence type ST198, the XbaI-pulsed-field gel electrophoresis cluster X1, and the presence of the Salmonella genomic island 1 variant SGI1-K. This clone was probably selected in 3 steps in Egypt during the 1990s and the early 2000s and has now spread to several countries in Africa and, more recently, in the Middle East. Poultry has been identified as a potential major vehicle for infection by this clone. Continued surveillance and appropriate control measures should be implemented by national and international authorities to limit the spread of this strain.

Quinolone-resistant Campylobacter infections: risk factors and clinical consequences.

We integrated data on quinolone and macrolide susceptibility patterns with epidemiologic and typing data from Campylobacter jejuni and C. coli infections in two Danish counties. The mean duration of illness was longer for 86 patients with quinolone-resistant C. jejuni infections (median 13.2 days) than for 381 patients with quinolone-sensitive C. jejuni infections (median 10.3 days, p = 0.001). Foreign travel, eating fresh poultry other than chicken and turkey, and swimming were associated with increased risk for quinolone-resistant C. jejuni infection. Eating fresh chicken (of presumably Danish origin) was associated with a decreased risk. Typing data showed an association between strains from retail food products and broiler chickens and quinolone-sensitive domestically acquired C. jejuni infections. An association between treatment with a fluoroquinolone before stool-specimen collection and having a quinolone-resistant C. jejuni infection was not observed.

Comparison of Genotypes and Serotypes of Campylobacter jejuni Isolated from Danish Wild Mammals and Birds and from Broiler Flocks and Humans

ABSTRACT The incidence of human infection with Campylobacter jejuni is increasing in most developed countries and the reason for this is largely unknown. Although poultry meat is considered to be a major source, it is evident that other reservoirs exist, possibly common to humans and poultry. Environmental sources are believed to be important reservoirs of Campylobacter infection in broiler chicken flocks. We investigated the potential importance of wildlife as a source of infection in commercial poultry flocks and in humans by comparing the serotype distributions, fla types, and macrorestriction profiles (MRPs) of C. jejuni isolates from different sources. The serotype distribution in wildlife was significantly different from the known distributions in broilers and humans. Considerable sero- and genotype diversity was found within the wildlife collection, although two major groups of isolates within serotype O:12 and the O:4 complex were found. Common clonal lines among wildlife, chicken, and/or human isolates were identified within serotype O:2 and the O:4 complex. However, MRPs of O:12 and O:38 strains isolated from wildlife and other sources indicated that some clonal lines propagated in a wide selection of animal species but were not detected in humans or broilers in this study. The applied typing methods successfully identified different clonal groups within a strain collection showing large genomic diversity. However, the relatively low number of wildlife strains with an inferred clonal relationship to human and chicken strains suggests that the importance of wildlife as a reservoir of infection is limited.

Tandem Repeat Analysis for Surveillance of Human Salmonella Typhimurium Infections

Multiple-locus variable-number tandem repeats analysis improves surveillance and outbreak investigations.

Evaluation of whole genome sequencing for outbreak detection of Salmonella enterica.

Salmonella enterica is a common cause of minor and large food borne outbreaks. To achieve successful and nearly ‘real-time’ monitoring and identification of outbreaks, reliable sub-typing is essential. Whole genome sequencing (WGS) shows great promises for using as a routine epidemiological typing tool. Here we evaluate WGS for typing of S. Typhimurium including different approaches for analyzing and comparing the data. A collection of 34 S. Typhimurium isolates was sequenced. This consisted of 18 isolates from six outbreaks and 16 epidemiologically unrelated background strains. In addition, 8 S. Enteritidis and 5 S. Derby were also sequenced and used for comparison. A number of different bioinformatics approaches were applied on the data; including pan-genome tree, k-mer tree, nucleotide difference tree and SNP tree. The outcome of each approach was evaluated in relation to the association of the isolates to specific outbreaks. The pan-genome tree clustered 65% of the S. Typhimurium isolates according to the pre-defined epidemiology, the k-mer tree 88%, the nucleotide difference tree 100% and the SNP tree 100% of the strains within S. Typhimurium. The resulting outcome of the four phylogenetic analyses were also compared to PFGE reveling that WGS typing achieved the greater performance than the traditional method. In conclusion, for S. Typhimurium, SNP analysis and nucleotide difference approach of WGS data seem to be the superior methods for epidemiological typing compared to other phylogenetic analytic approaches that may be used on WGS. These approaches were also superior to the more classical typing method, PFGE. Our study also indicates that WGS alone is insufficient to determine whether strains are related or un-related to outbreaks. This still requires the combination of epidemiological data and whole genome sequencing results.

Verocytotoxin-Producing Escherichia coli in Wild Birds and Rodents in Close Proximity to Farms

ABSTRACT Wild animals living close to cattle and pig farms (four each) were examined for verocytotoxin-producing Escherichia coli (VTEC; also known as Shiga toxin-producing E. coli). The prevalence of VTEC among the 260 samples from wild animals was generally low. However, VTEC isolates from a starling (Sturnus vulgaris) and a Norway rat (Rattus norvegicus) were identical to cattle isolates from the corresponding farms with respect to serotype, virulence profile, and pulsed-field gel electrophoresis type. This study shows that wild birds and rodents may become infected from farm animals or vice versa, suggesting a possible role in VTEC transmission.

Outbreak of Non-O157 Shiga Toxin-Producing Escherichia coli Infection from Consumption of Beef Sausage

We describe an outbreak of Shiga toxin-producing Escherichia coli O26:H11 infection in 20 patients (median age, 2 years). The source of the infection was an organic fermented beef sausage. The source was discovered by using credit card information to obtain and compare customer transaction records from the computer systems of supermarkets.

Packed with Salmonella--investigation of an international outbreak of Salmonella Senftenberg infection linked to contamination of prepacked basil in 2007.

Salmonella Senftenberg is uncommon in the United Kingdom. In January-June 2007, the Health Protection Agency reported on 55 primary human cases of Salmonella Senftenberg in England and Wales. In May 2007, fresh basil sold in the United Kingdom was found to be contaminated with Salmonella Senftenberg. We launched an investigation to elucidate the cause of this outbreak. Isolates were examined using plasmid profiling and pulsed-field gel electrophoresis, and the outbreak strain (SSFTXB.0014) was identified. We enquired via Enter-net whether other countries had isolated the outbreak strain, analyzed samples of fresh herbs from U.K. retailers, and interviewed patients on food history. Thirty-two patient-cases were referred to this outbreak in England and Wales. Onsets of illness occurred between 5 March and 6 June 2007. Fifty-six percent of patient-cases were females and 90% adults (>20 years old); three were admitted to hospital as a result of Salmonella infection. Scotland, Denmark, the Netherlands, and the United States reported on 19 cases of Salmonella Senftenberg infection presenting with the outbreak strain since January 2007. Eight samples of prepacked fresh basil imported from Israel tested positive with the same strain. A minority of patients could recall the consumption of basil before illness, and some reported consumption of products where basil was a likely ingredient. Environmental investigations in Israel did not identify the contamination source. Microbiological evidence suggested an association between contamination of fresh basil and the cases of Salmonella Senftenberg infection, leading to withdrawal of basil from all potentially affected batches from the U.K. market.