Due to the potential of enterohemorrhagic Escherichia coli (EHEC) serogroup O157 to cause large food borne outbreaks, national and international surveillance is necessary.
For developing an effective method of molecular surveillance, a conventional method, multilocus variable-number tandem-repeat analysis (MLVA), and whole-genome sequencing (WGS) analysis were compared. WGS of 369 isolates of EHEC O157 belonging to 7 major MLVA types and their relatives were subjected to comprehensive in silico typing, core genome single nucleotide polymorphism (cgSNP), and core genome multilocus sequence typing (cgMLST) analyses. The typing resolution was the highest in cgSNP analysis. However, determination of the sequence of the mismatch repair protein gene mutS is necessary because spontaneous deletion of the gene could lead to a hypermutator phenotype. MLVA had sufficient typing resolution for a short-term outbreak investigation and had advantages in rapidity and high throughput. cgMLST showed less typing resolution than cgSNP, but it is less time-consuming and does not require as much computer power. Therefore, cgMLST is suitable for comparisons using large data sets (e.g., international comparison using public databases). In conclusion, screening using MLVA followed by cgMLST and cgSNP analyses would provide the highest typing resolution and improve the accuracy and cost-effectiveness of EHEC O157 surveillance.
IMPORTANCE Intensive surveillance for enterohemorrhagic Escherichia coli (EHEC) serogroup O157 is important to detect outbreaks and to prevent the spread of the bacterium. Recent advances in sequencing technology made molecular surveillance using whole-genome sequence (WGS) realistic. To develop rapid, high-throughput, and cost-effective typing methods for real-time surveillance, typing resolution of WGS and a conventional typing method, multilocus variable-number tandem-repeat analysis (MLVA), was evaluated. Nation-level systematic comparison of MLVA, core genome single nucleotide polymorphism (cgSNP), and core genome multilocus sequence typing (cgMLST) indicated that a combination of WGS and MLVA is a realistic approach to improve EHEC O157 surveillance.
Effective surveillance using multilocus variable-number tandem-repeat analysis and whole-genome sequencing for enterohemorrhagic Escherichia coli O157
Applied and Environmental Microbiology
Kenichi Lee, Hidemasa Izumiya, Sunao Iyoda, Makoto Ohnishi and EHEC Working Group
As of August 18, 2019, there have been 7 confirmed cases of Listeria monocytogenes linked to Rosemount brand cooked diced chicken in British Columbia (1), Manitoba (1) and Ontario (5). Individuals became sick between November 2017 and June 2019. Six individuals have been hospitalized. Individuals who became ill are between 51 and 97 years of age. The majority of cases (86%) are female.
The collaborative outbreak investigation was initiated because of an increase of Listeria illnesses that were reported in June 2019. Through the use of whole genome sequencing, two Listeria illnesses from November 2017 were identified to have the same genetic strain as the illnesses that occurred between April and June 2019.
It is possible that more recent illnesses may be reported in the outbreak because of the delay between when a person becomes ill and when the illness is reported to public health officials. In national Listeria monocytogenes outbreak investigations, the case reporting delay is usually between 4 and 6 weeks.
If you have Rosemount brand cooked diced chicken meat 13mm – ½” (#16305), packdate – 01/21/2019 in your food establishment, do not eat the product or serve it to others
Secure the product and any foods made with the product in a plastic bag, throw it out and wash your hands with soapy water.
In 1999, I gave a talk to hundreds of farm leaders in Ottawa and told them that DNA fingerprinting – via PulseNet – would revolutionize foodborne illness outbreak investigations and that farmers better be prepared (the pic is from a 2003 awards ceremony where I was acknowledged for my outreach and extension efforts, the hair was fabulous).
Twenty years later and whole genome sequencing is even further piecing together disparate outbreaks.
Joanie Stiers of Farm Flavor writes that Michigan’s laboratory toolbox now includes whole-genome sequencing, allowing public health officials to stop the spread of foodborne illness faster than ever.
Since January of 2017, the Michigan Department of Agriculture and Rural Development (MDARD) has actively used whole-genome sequencing to precisely identify illness-causing pathogens and defend against widespread outbreaks of foodborne diseases.
“With food now being distributed worldwide, illness can be spread from anywhere in the world,” says Ted Gatesy, laboratory manager of the microbiology section at Geagley Lab, which houses the whole-genome sequencing. “Using whole-genome sequencing, an illness can be tracked, for the most part, to the point in the food chain where it originated.
During summer 2016, Norway observed an increase in Salmonella enterica subsp. enterica serovar Chester cases among travellers to Greece.
Our aim was to investigate genetic relatedness of S. Chester for surveillance and outbreak detection by core genome multilocus sequence typing (cgMLST) and compare the results to genome mapping.
We included S. Chester isolates from 51 cases of salmonellosis between 2000 and 2016. Paired-end sequencing (2 × 250 bp) was performed on Illumina MiSeq. Genetic relatedness by cgMLST for Salmonellaenterica subsp. enterica, including 3,002 genes and seven housekeeping genes, was compared by reference genome mapping with CSI Phylogeny version 1.4 and conventional MLST.
Confirmed travel history was available for 80% of included cases, to Europe (n = 13), Asia (n = 12) and Africa (n = 16). Isolates were distributed into four phylogenetic clusters corresponding to geographical regions. Sequence type (ST) ST411 and a single-locus variant ST5260 (n = 17) were primarily acquired in southern Europe, ST1954 (n = 15) in Africa, ST343 (n = 11) and ST2063 (n = 8) primarily in Asia. Part of the European cluster was further divided into a Greek (n = 10) and a Cypriot (n = 4) cluster. All isolates in the African cluster displayed resistance to ≥ 1 class of antimicrobials, while resistance was rare in the other clusters.
Whole genome sequencing of S. Chester in Norway showed four geographically distinct clusters, with a possible outbreak occurring during summer 2016 related to Greece. We recommend public health institutes to implement cgMLST-based real-time Salmonella enterica surveillance for early and accurate detection of future outbreaks and further development of cluster cut-offs.
Whole genome sequencing of Salmonella Chester reveals geographically distinct clusters, Norway, 2000 to 2016
Siira Lotta, Naseer Umaer, Alfsnes Kristian, Hermansen Nils Olav, Lange Heidi, Brandal Lin T. Whole genome sequencing of Salmonella Chester reveals geographically distinct clusters, Norway, 2000 to 2016. Euro Surveill. 2019;24(4):pii=1800186. https://doi.org/10.2807/1560-7917.ES.2019.24.4.1800186
A multi-country outbreak of 12 listeriosis cases caused by Listeria monocytogenes sequence type (ST) 8 has been identified through whole genome sequencing (WGS) analysis in three EU/EEA countries: Denmark (6 cases), Germany (5) and France (1).
Four of these cases have died due to or with the disease. It is likely that the extent of this outbreak has been underestimated since the outbreak was identified through sequencing and only a subset of the EU/EEA countries routinely use this advanced technique to characterise L. monocytogenes isolates.
The first case was sampled in October 2015 in Denmark and the most recent case was reported in May 2018 in Germany. In August 2017, Denmark identified the first cluster of cases, which was investigated and linked to the consumption of ready-to-eat cold-smoked salmon produced in Poland. Control measures were implemented and the Member States and competent authorities were informed.
In October 2017, France reported the identification of a matching L. monocytogenes strain in food isolates from marinated salmon originating from the same Polish processing company as identified in the Danish outbreak investigation. This supports the hypothesis that contamination may have occurred at the processing company in Poland. However, due to the lack of WGS data on the isolates found in the environmental and food samples taken at the Polish processing plant, it is not possible at present to confirm the contamination with the L. monocytogenes ST8 outbreak strain at the suspected Polish plant. Moreover, until detailed information on the Norwegian primary producers of the salmon used in the contaminated batches is reported and assessed, possible contamination at primary production level cannot be excluded either.
Although control measures were implemented following the Danish outbreak investigation in September 2017, the identification of the same strain in a salmon product in France and a new human case in Germany suggest that the source of contamination is still active and contaminated products have been distributed to other EU countries than Denmark.
Until the source of infection has been eliminated, new invasive listeriosis cases may still occur. Pregnant women, the elderly and immunocompromised individuals are at increased risk of invasive listeriosis, which is associated with severe clinical course and potentially death.
In Denmark, on 23 August 2017, Statens Serum Institut (SSI) identified a genetic cluster of four human Listeria monocytogenes sequence type (ST) 8 isolates by core genome multilocus sequence typing (cgMLST) [1]. The allele calling was performed in BioNumerics (v7.6.2, Applied Maths, Belgium). We initiated an epidemiological investigation and notified the Danish Central Outbreak Management Group (collaboration between the Danish Veterinary and Food Administration (DVFA), the National Food Institute at the Technical University of Denmark (DTU) and SSI). On 25 August, two additional human isolates were found to belong to the same genetic cluster.
A confirmed case was defined as a person clinically diagnosed with listeriosis after 1 January 2017 with laboratory-confirmed L. monocytogenes ST8 clustering using cgMLST (≤ 5 allelic distance, single linkage). Cases diagnosed before 1 January 2017 with an isolate belonging to this cluster were defined as probable cases.
As of 25 August 2017, the genetic cluster comprised six cases; five confirmed and one probable. The age of the cases ranged from 59 to 96 years (median 80 years) and four were women. All patients had underlying illness and no travel history. One patient died within 30 days of diagnosis. Epidemiological investigations including a standard questionnaire on exposures showed that all five confirmed cases had consumed cold-smoked and/or cured salmon in the 30 days before disease onset. Four cases had bought the salmon in retail chain X. No other food-item was reported as consumed in high frequencies among cases. Epidemiological follow-up for the probable case did not include information on fish consumption.
On 29 August 2017, a comparison between the human outbreak isolates and 16 L. monocytogenes ST8 food- and environmental isolates identified in Denmark from 2014 to August 2017 showed that the human isolates clustered with a food isolate from cold-smoked salmon, cut and packaged at company Y in Poland (zero to two allelic differences using cgMLST). L. monocytogenes had been detected on 31 July 2017 at levels of 110 CFU/g (threshold: 100 CFU/g) at the end of shelf life. The product was widely sold in Denmark and had been sampled by the DVFA in retail chain X, as part of a consumer exposure survey (i.e. analyses project on retail packages). Because the L. monocytogenes concentration had been just above the accepted limit and found at the end of the product shelf life a recall of this batch was not conducted. However, due to the positive finding, follow-up sampling had been performed on the 9 and 10 August 2017 from the central storage unit of retail chain X. L. monocytogenes had been isolated from two batches analysed before end of shelf life. In one sample from the same batches, which was also analysed at the end of the shelf life, on 28 August 2017 a L. monocytogenes level of 240 CFU/g was found. Isolates from the follow-up samples had zero to four allelic differences to the human outbreak isolates using cgMLST.
The human outbreak sequences were also compared to all L. monocytogenes ST8 genomes derived from clinical samples in Denmark from 2012 onwards. Although ST8 genomes from Danish patients in the period 2012–2017 showed high diversity, the outbreak isolates clearly formed a distinct cgMLST cluster with 16 allelic differences to the nearest isolates outside the genetic outbreak cluster and a maximum of nine allelic differences within the cluster (Figure 2a). We investigated the relatedness of outbreak isolates further by single-nucleotide polymorphisms (SNP) analysis performed by both SSI and DTU using two analysis pipelines: Northern Arizona SNP Pipeline (NASP) [2] and CSI Phylogeny version 1.4 from Center for Genomics Epidemiology (CGE), DTU [3] leading to the same conclusion.
On 30 August 2017, DVFA advised retail chain X to recall all cold-smoked salmon produced at company Y. This advice was based on the elevated number of L. monocytogenes (240 CFU/g) found in the product at the end of shelf-life and the link to the outbreak. Retail chain X voluntarily recalled both cold-smoked and cured salmon produced at company Y. As part of the recall procedure, retail chain X informed company Y on the situation. Information from company Y, provided by the Polish food authorities via the European Union Rapid Alert System for Food and Feed (RASFF), showed that the implicated batches were exclusively sold via retail chain X and only in Denmark.
The French National Reference Centre (NRC) for Listeria (Institut Pasteur, Paris), compared the sequences of the Danish human isolates against its database, using cgMLST as previously described [1,4]. A human isolate from a French resident belonged to the same cluster (L2-SL8-ST8-CT771) as the Danish isolates. This French probable case, a female patient in her mid-80s, was diagnosed in June 2016. Epidemiological investigations carried out by Santé Publique France were inconclusive, since food consumption history was not available at the time of diagnosis nor could information on travel to Denmark be retrieved, as the person had since died.
On 6 September 2017, an official control by the Ministry of Economy was carried out at a French retailer where a kosher chilled cured salmon was sampled for analysis. The sample was contaminated with L. monocytogenes at the level of 460 CFU/g and the salmon producer was company Y. An isolate was sent to the French NRC for typing and showed to belong to the same cgMLST type as the Danish outbreak. Further investigations on the food product confirmed that it had not been further processed after production in Poland. The product was recalled and no human cases were linked to its consumption as of beginning of December 2017.
The other nine countries that replied to the EPIS-FWD UI-426 notification (Austria, Finland, Germany, Luxembourg, the Netherlands, Norway, Slovenia, Sweden, United Kingdom) did not report any human or food isolates linked to the Danish outbreak. However, after submission of this report, at the end of November, we were informed through EPIS about three genetically linked human isolates in Germany.
Discussion
Here we report on a listeriosis outbreak and highlight the value of rapidly comparing the genomes of human and food/environmental isolates at the national and international levels.
The fact that the contaminated salmon products identified in Denmark and France were from different batches suggests environmental contamination possibly at the production facility at company Y. It is too early to assess whether any measures taken at company Y have been effective in controlling the outbreak. However, experiences from previous investigations suggest that once L. monocytogenes is detected in one product, the whole production site should be subject to a thorough inspection, and sampling with special attention to all the possible contamination/cross contamination issues before implementing corrective measures [5,6]. Moreover, the risk for L. monocytogenes persistent strains in the production environment requires the close monitoring for several years to ensure the elimination of these [7,8].
Since WGS was introduced for routine surveillance in Denmark, a number of listeriosis outbreaks have been detected and solved, including outbreaks involving cold-smoked ready-to-eat sliced fish products [5]. The present investigation further reinforces the suspicion that ready-to-eat fish products are important sources of L. monocytogenes infections in Denmark, as well as in other countries.
Though only involving a low number of isolates, WGS L. monocytogenes surveillance and communication between countries allowed us to detect and rapidly solve this salmon-associated outbreak, leading to food product recall in two European countries. Compared with previous typing methods, WGS has a higher discriminatory power and the ability to determine genetic distance between isolates. The introduction of WGS for surveillance of food-borne infections has shown that it improves outbreak detection and facilitates outbreak investigations and likely helps reduce the number of infections [4,9-16]. The EPIS-FWD communication platforms allowed for the communication to link cases across borders. However, currently cross-border outbreaks are only detected when case numbers in at least one country exceed normal levels and are notified internationally. Therefore, a possible future system for easy exchange of and comparison of WGS data, e.g. by the use of an agreed cgMLST nomenclature, across borders will enable the identification of more dispersed outbreaks as well as cross-border links between food samples and human infections. This report highlights that by the application of cross-disciplinary and real-time cross-border comparison of WGS data, L. monocytogenes infections can be prevented and thereby providing safer food for at-risk groups such as the elderly, immunodeficient individuals and pregnant women.
Cross-border outbreak of listeriosis caused by cold-smoked salmon, revealed by integrated surveillance and whole genome sequencing (WGS), Denmark and France, 2015 to 2017
Schjørring Susanne, Gillesberg Lassen Sofie , Jensen Tenna, Moura Alexandra, Kjeldgaard Jette S, Müller Luise, Thielke Stine, Leclercq Alexandre, Maury Mylene M, Tourdjman Mathieu, Donguy Marie-Pierre, Lecuit Marc, Ethelberg Steen, Nielsen Eva M. Cross-border outbreak of listeriosis caused by cold-smoked salmon, revealed by integrated surveillance and whole genome sequencing (WGS), Denmark and France, 2015 to 2017. Euro Surveill. 2017;22(50):pii=17-00762. https://doi.org/10.2807/1560-7917.ES.2017.22.50.17-00762
Building on their work with whole genome sequencing and eggs – because there’s a lot of outbreaks of Salmonella in eggs — a group of Australian researchers have reported on seven outbreaks of Salmonella Typhimurium multilocus variable-number tandem-repeat analysis (MLVA) 03-26-13-08-523 (European convention 2-24-12-7-0212) in three Australian states and territories investigated between November 2015 and March 2016.
We identified a common egg grading facility in five of the outbreaks. While no Salmonella Typhimurium was detected at the grading facility and eggs could not be traced back to a particular farm, whole genome sequencing (WGS) of isolates from cases from all seven outbreaks indicated a common source. WGS was able to provide higher discriminatory power than MLVA and will likely link more Salmonella Typhimurium cases between states and territories in the future. National harmonization of Salmonella surveillance is important for effective implementation of WGS for Salmonella outbreak investigations.
Seven Salmonella Typhimurium outbreaks in Australia linked by trace-back and whole genome sequencing
Foodborne Pathogens and Disease, March, 2018, 10.1089/fpd.2017.2353
Laura Ford Qinning Wang Russell Stafford,Kelly-Anne Ressler, Sophie Norton, Craig Shadbolt, Kirsty Hope, Neil Franklin, Radomir Krsteski, Adrienne Carswell,Glen P. Carter, Torsten Seemann,Peter Howard, Mary Valcanis,10 Cristina Fabiola Sotomayor Castillo, John Bates, Kathryn Glass,Deborah A. Williamson, Vitali Sintchenko, Benjamin P. Howden and Martyn D. Kirk1
To compare WGS-based approaches with conventional typing for Salmonella surveillance, we performed concurrent WGS and multilocus variable-number tandem-repeat analysis (MLVA) of Salmonella Typhimurium isolates from the Australian Capital Territory (ACT) for a period of 5 months. We exchanged data via a central shared virtual machine and performed comparative genomic analyses. Epidemiological evidence was integrated with WGS-derived data to identify related isolates and sources of infection, and we compared WGS data for surveillance with findings from MLVA typing.
We found that WGS data combined with epidemiological data linked an additional 9% of isolates to at least one other isolate in the study in contrast to MLVA and epidemiological data, and 19% more isolates than epidemiological data alone. Analysis of risk factors showed that in one WGS-defined cluster, human cases had higher odds of purchasing a single egg brand. While WGS was more sensitive and specific than conventional typing methods, we identified barriers to uptake of genomic surveillance around complexity of reporting of WGS results, timeliness, acceptability, and stability.
In conclusion, WGS offers higher resolution of Salmonella Typhimurium laboratory surveillance than existing methods and can provide further evidence on sources of infection in case and outbreak investigations for public health action. However, there are several challenges that need to be addressed for effective implementation of genomic surveillance in Australia.
Incorporating whole-genome sequencing into public health surveillance: Lessons from prospective sequencing of salmonella typhimurium in Australia
16 January 2018
Foodborne Pathogens and Disease
Laura Ford, Glen Carter, Qinning Wang, Torsten eemann, Vitali Sintchenko, Kathryn Glass, Deborah Williamson, Peter Howard, Mary Valcanis, Cristina Castillo, Michelle Sait, Benjamin Howden, and Martyn Kirk
The Ohio Department of Health, several other states, the U.S. Centers for Disease Control, and USDA-APHIS are investigating a multistate outbreak of human Campylobacter infections linked to puppies sold through Petland stores.
Investigators are looking for the source of infections in people and puppies so they can recommend how to stop the outbreak and prevent more illnesses in order to protect human and animal health.
As of September 11, 2017, the outbreak includes 39 cases in 7 states (Florida, Kansas, Missouri, Ohio, Pennsylvania, Tennessee, and Wisconsin).
Illnesses began on dates ranging from September 15, 2016 through August 12, 2017. The most recent illness was reported on September 1, 2017.
Ill people range in age from <1 year to 64 years, with a median age of 22 years; 28 (72%) are female; and 9 (23%) report being hospitalized. No deaths have been reported.
Epidemiologic and laboratory findings have linked the outbreak to contact with puppies sold through Petland stores. Among the 39 ill people, 12 are Petland employees from 4 states and 27 either recently purchased a puppy at Petland, visited a Petland, or visited or live in a home with a puppy sold through Petland before illness began.
Whole genome sequencing showed samples of Campylobacter isolated from the stool of puppies sold through Petland in Florida were closely related to Campylobacter isolated from the stool of an ill person in Ohio. Additional laboratory results from people and dogs are pending.
Regardless of where they are from, any puppies and dogs may carry Campylobacter germs.
In 1993, I thought the consummation of Hollywood and Nashville was complete when starlet Julia Roberts wed country music’s ugly duckling, Lyle Lovett.
They divorced less than two years later.
As the NHL Stanley Cup playoffs progressed, the last-seeded Nashville Predators demolished foe-after-foe, with star couple captain Mike Fischer and partner Carrie Underwood paving the way for another coupling of the weirdness and greatness that is America: Nashville and Smashville.
But I don’t think that Hank do it like that.
It didn’t happen, as Nashville finally lost to Pittsburgh in 6-games to close out a grueling National Hockey League season.
I write this while watching Sorenne and Amy on the ice, taking extra skating lessons, in the sub-tropics of Brisbane, as likely a hockey hotspot as Nashville.
Amy is happy Pittsburgh won because, she hates country music.
But baby … Lyle isn’t country, he’s something different.
I’ve been to Nashville several times, hung out on Music Row, hung out a Titans tailgate, and saw Lyle Lovett one night and John Prine the next at the Ryman Auditorium, The Mother Church of Country Music.
Now that my Nashville Predators have lost the Stanley Cup in a valiant, country-heartbreak ballad to Pittsburgh, I return to the more mundane mattes of foodborne illness.
PulseNet International advocates for public health institutes and laboratories around the world to move together towards the use of whole genome sequencing (WGS) to improve detection of and response to foodborne illnesses and outbreaks in the latest edition of Eurosurveillance.
PulseNet International is a global network of public health laboratory networks, dedicated to bacterial foodborne disease surveillance. The network is comprised of the national and regional laboratory networks of USA, Canada, Latin America and the Caribbean, Europe, Africa, the Middle-East and Asia Pacific.
The European Centre for Disease Prevention and Control (ECDC) manages the EU/EEA food- and waterborne diseases and zoonoses network of public health institutes and laboratories, which work to ensure comparability of data and further ties to the global health community.
Mike Catchpole, Chief Scientist at ECDC says, “it is important for all partners worldwide to continue to work together towards the implementation and standardised analysis of whole genome sequencing.”
The article also states that a global standard method for primary sequence data analysis based on whole genome Multiple Locus Sequence Typing (wgMLST) and derived public nomenclature will be adopted.
This will facilitate the sharing of information within regional and global public health laboratory networks, increasing efficiency and enabling data to be compared across countries in real-time which is currently not the case. This is especially important due to international travel and trade.
Common steps for validation studies, development of standardised protocols, quality assurance programmes and nomenclature have been agreed.
Pulsenet international: Vision for the implementation of whole genome sequencing (WGS) for global food-borne disease surveillance
Eurosurveillance, vol. 22, issue, 23, 08 June 2017, C Nadon , I Van Walle, P Gerner-Smidt, J Campos, Chinen, J Concepcion-Acevedo, B Gilpin, AM Smith, KM Kam, E Perez, E Trees, K Kubota, J Takkinen, EM Nielsen, H Carleton, FWD-NEXT Expert Panel, DOI: http://dx.doi.org/10.2807/1560-7917.ES.2017.22.23.30544
PulseNet International is a global network dedicated to laboratory-based surveillance for food-borne diseases. The network comprises the national and regional laboratory networks of Africa, Asia Pacific, Canada, Europe, Latin America and the Caribbean, the Middle East, and the United States. The PulseNet International vision is the standardised use of whole genome sequencing (WGS) to identify and subtype food-borne bacterial pathogens worldwide, replacing traditional methods to strengthen preparedness and response, reduce global social and economic disease burden, and save lives. To meet the needs of real-time surveillance, the PulseNet International network will standardise subtyping via WGS using whole genome multilocus sequence typing (wgMLST), which delivers sufficiently high resolution and epidemiological concordance, plus unambiguous nomenclature for the purposes of surveillance. Standardised protocols, validation studies, quality control programmes, database and nomenclature development, and training should support the implementation and decentralisation of WGS. Ideally, WGS data collected for surveillance purposes should be publicly available, in real time where possible, respecting data protection policies. WGS data are suitable for surveillance and outbreak purposes and for answering scientific questions pertaining to source attribution, antimicrobial resistance, transmission patterns, and virulence, which will further enable the protection and improvement of public health with respect to food-borne disease.
Whole genome sequencing (WGS) for food-borne pathogen surveillance and control- Taking the pulse
Next-generation sequencing (NGS) is transforming microbiology [1]. With the increased accessibility and decrease in the costs of sequencing and the optimisation of the ‘wet laboratory’ components of NGS i.e. the quality and throughput of DNA extraction, library preparation and sequencing reactions, whole genome sequencing (WGS) of bacterial isolates is rapidly revolutionising clinical and public health microbiology. WGS is a ‘disruptive technology’ that has the potential to become a one-stop-shop for routine bacterial analysis. By replacing multiple parallel steps in the microbiology diagnostic cycle, which currently involves traditional and molecular methods, it achieves accurate and speedy species identification, inference of antimicrobial susceptibility and virulence and high-resolution subtyping [2].
Typing of food-borne pathogens was one of the earliest applications of WGS [3] and proof-of-concept has been demonstrated for the superiority of WGS over traditional typing methods such as pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST), for a range of high priority food-borne pathogens, including Salmonella enterica, Listeria monocytogenes, Campylobacter species and Shiga-toxin producing Escherichia coli [4]. Applications of WGS include the investigation of food-related outbreaks and surveillance to delineate the local, regional and global genomic epidemiology of pathogens and to attribute the infection source. WGS thus supports risk assessment and guides interventions for prevention and control of infections.
A growing number of (public health microbiology) laboratories and governmental agencies employ WGS in their routine practice and food-borne pathogen surveillance and even more are expected to enter this field in the near future. Thus the maturation of food-borne pathogen surveillance into the WGS era is very timely.
In order for WGS to be adopted as the new gold standard for tracking of food-borne pathogens, a key element of food-borne disease control, there is a need for robust, standardised, portable and scalable methods for analysing WGS data. However, the notable diversity of bioinformatics tools and approaches used for bacterial WGS to date, as evident from a recent survey by the Global Microbial Identifier project [5], creates a tremendous challenge for harmonising surveillance and investigation of food-borne illness, especially across geographical borders and different sectors. Calling variants based on analysis of single nt polymorphisms (SNPs) as it is being done in many food-borne outbreak investigations, offers maximal resolution and discriminatory power but is very difficult to standardise. Therefore, approaches based on gene-by-gene analyses, collectively referred to as ‘extended MLST’, such as core genome (cg) or whole genome (wg)MLST may be advantageous [6], and have been advocated in other public health settings, such as Legionnaires’ disease control [7].
PulseNet was established in the United States (US) more than 20 years ago as a laboratory network for molecular epidemiology based on standardised PFGE analysis and later expanded globally. PulseNet has been successful in engaging many players in the field of food safety on a global scale and in creating a platform for data sharing and comparison of clinical, veterinary and food isolates in over 80 countries and it has a proven track-record in supporting molecular surveillance [8]. Nevertheless, some issues remained unresolved such as creation and implementation of a global nomenclature, which is important for communicating molecular epidemiology results, both scientifically as well as operationally.
In this issue of Eurosurveillance, an article by Nadon et al. [9] describes the next generation of PulseNet International, which is evolving into harnessing WGS. This initiative represents a wide collaboration between many leading agencies and stakeholders in this area, including the US Centers for Disease Control and Prevention (CDC), the European Centre for Disease Prevention and Control (ECDC) and the Public Health Agency Canada (PHAC), just to name a few. The authors illustrate the technical and practical aspects of adapting the network. Notably, PulseNet International has chosen an extended MLST approach, specifically, wgMLST, as its default phylogenetic analysis tool, which should underpin a standardised and efficient nomenclature-based system. Different technical and practical aspects are reviewed and discussed, mainly focusing on information technology (IT) and bioinformatics aspects (data storage, computing power, nomenclature, data sharing), methods for validation and quality control/quality assurance. Nadon et al. highlight complexities surrounding the implementation of WGS for food-borne disease surveillance, with respect to readiness at individual country and regional levels and delineate how PulseNet plans to address these.
The evolution of PulseNet International is very encouraging and will reinforce the use of NGS in the area of food safety. That said, challenges remain that need to be addressed by the public health community. There is a need for user-friendly bioinformatics solutions that will enable automated analysis of bacterial genomes by non-experts in bioinformatics to extract valuable information in a time-efficient manner. Such solutions should offer as much backwards compatibility as possible with current typing methods since the global transition to WGS is expected to be gradual. It should also offer an efficient strain/allele nomenclature that facilitates inter-laboratory work. Moreover, bioinformatics solutions should also factor in the developments in the field of DNA sequencing, particularly long-read single molecule sequencing platforms and portable sequencing devices which are increasingly being used. While WGS of food-borne pathogens has now become the new gold standard for food-borne pathogen typing, other techniques such as strain typing and characterisation using proteomics (particularly matrix-assisted laser desorption/ionisation (MALDI) time-of-flight (TOF) mass spectroscopy) or DNA arrays are rapidly evolving and should be carefully evaluated [10]. The field of metagenomics is also rapidly advancing and culture-independent microbiology, enabling genomic analysis of pathogens directly from sequenced clinical or environmental samples (as opposed to cultured isolates), is just around the corner [11]. When laying the foundations for global food pathogen surveillance networks for the coming years, we need to be mindful of such future developments.
Different from current protocols in which only typing results are shared, the transition to genome-based surveillance inevitably involves the sharing of complete sequence data. This has many implications, not only with respect to data storage, analysis and sharing infrastructures, but also aspects such as data ownership, privacy and transparency, pertaining to both genomic sequences and the related metadata. These issues should be proactively addressed in order to provide reassurance concerning data protection and create flexible solutions that will facilitate the timely sharing of public health data by as many partners as possible.
Finally, the transition to WGS-based surveillance needs to ensure sufficient quality is maintained in order to meet national and international regulatory requirements. Nadon et al. rightfully emphasise in their paper, the importance of validation, quality control and standardisation. One major aspect in making this transition and that needs to be considered is the human factor. The successful implementation of WGS-based surveillance on a global scale requires careful planning, building of capacity and training of public health and microbiology personnel to develop local readiness, especially in limited resource settings. Care should be taken to address the ‘softer’ issues, including possible cultural, political and cross-sector barriers, which together with economical, management and operational aspects could greatly influence the successful implementation of WGS.
This is a fascinating time for public health microbiology, and initiatives such as the integration of WGS as proposed by PulseNet International, are central for leveraging recent technological advancements for the benefit of public health surveillance.
For developing an effective method of molecular surveillance, a conventional method, multilocus variable-number tandem-repeat analysis (MLVA), and whole-genome sequencing (WGS) analysis were compared. WGS of 369 isolates of EHEC O157 belonging to 7 major MLVA types and their relatives were subjected to comprehensive in silico typing, core genome single nucleotide polymorphism (cgSNP), and core genome multilocus sequence typing (cgMLST) analyses. The typing resolution was the highest in cgSNP analysis. However, determination of the sequence of the mismatch repair protein gene mutS is necessary because spontaneous deletion of the gene could lead to a hypermutator phenotype. MLVA had sufficient typing resolution for a short-term outbreak investigation and had advantages in rapidity and high throughput. cgMLST showed less typing resolution than cgSNP, but it is less time-consuming and does not require as much computer power. Therefore, cgMLST is suitable for comparisons using large data sets (e.g., international comparison using public databases). In conclusion, screening using MLVA followed by cgMLST and cgSNP analyses would provide the highest typing resolution and improve the accuracy and cost-effectiveness of EHEC O157 surveillance.
