November 2015

Detection and Molecular Epidemiology ofFoodborne and Animal Pathogens
IZSLER Parma laboratory adds the MiSeq® System to perform pathogen testing, epidemiology,
and genetic screening studies efficiently.

Introduction
The city of Parma is located in north central Italy’s beautiful Po Rivervalley. The fertile lower valley is often referred to as “food valley”, andis home to several gastronomic delights, including Prosciutto di Parmaham, Parmigiano-Reggiano cheese, as well as several international
food companies. It’s no wonder that Parma is the seat of theEuropean Food Safety Authority (EFSA) and a laboratory of the Istituto
Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna(IZSLER) that is committed to animal health and food safety.
As the veterinarian in charge of risk assessment at IZSLER and Headof IZSLER Parma, Stefano Pongolini, DVM, oversees a laboratory
team of 20 researchers and laboratory technologists, focusing on
the study, diagnosis, and prophylaxis of animal diseases and foodmicrobiology. With the recent addition of next-generation sequencing(NGS) to their analysis tool set, IZSLER Parma is performing viral andbacterial testing more efficiently, and providing services to a larger
customer population.
iCommunity spoke with Dr. Pongolini about the IZSLER Parmalaboratory, the testing services it provides, and how the MiSeq
System is enabling them to track foodborne illnesses andpathogens effectively.
Q: What types of services does IZSLER Parma provide to agri-foodbusinesses and how does it support public health?
Stefano Pongolini (SP): IZSLER is an official Institute of Veterinary
Public Health and operates at a regional level within the NationalHealth Service of Italy. We provide diagnostic and testing services for
2 economically important regions, Lombardy and Emilia-Romagna,
which represent about 23% of the Italian population and 30% of theGNP. Our routine testing activities serve the Regional Health Authorities
and local businesses. More research-oriented activities, such as thestudies we conduct using NGS, are offered to a broader group ofclients within and outside the region.

tracking human cases of foodborne diseases. The World Organization
for Animal Health (OIE) is also represented here with the Reference
Laboratory for Swine Influenza.
Q: What types of pathogen testing do you perform?
SP: There are many viruses and bacteria that we test for at thecenter, some that cause animal disease, and others that contributeto foodborne human illness and disease. We offer testing for several
swine viruses, including swine influenza virus, and porcine reproductive
and respiratory syndrome virus. We also can screen for various bovineviruses, including bovine viral diarrhea virus, bovine syncytial virus,
bovine rotavirus and coronavirus, and bovine rhinotracheitis virus.
In addition, we test for Hepatitis A virus and were able to contributeto the tracking of a 2013 HAV outbreak in Europe.1 We also test forflaviviruses, which include West Nile and the Usutu virus (USUV) thathave recently been on the rise in Europe.
The diagnosis and surveillance of bacterial pathogens encompasses
the full spectrum of animal and foodborne pathogens. These include
Escherichia coli, as well as bacteria in the Salmonella, Klebsiella,
Clostridium, Listeria, Staphylococcus, Streptococcus, Pseudomonas,
Pasteurella, Actinobacillus, and Mycoplasma genera. Because this is
an important milk and cheese producing region, we’re involved in thediagnosis of bovine mastitis, testing over 20,000 milk samples annuallyfor bacteria associated with the infection.

Our services can be grouped into 5 main areas: 1) animal diseasediagnostic testing, including zoonoses; 2) official controls on foodand feedstuffs; 3) epidemiological and analytical support to healthauthorities for the design and management of official prevention
and eradication plans; 4) monitoring and surveillance of animal andzoonotic diseases for foodborne diseases; and 5) applied and basic
research in infectious diseases.
IZSLER Parma is also the home of 2 reference centers. The Regional
ENTER-NET Reference Center runs the Enteric-Pathogens (foodborne)
surveillance system of the Emilia-Romagna region, covering a total
population of about 5,000,000 individuals. This surveillance includes

For Research Use Only. Not for use in diagnostic procedures.

Dr. Stefano Pongolini is the veterinarian in charge of risk
assessment at IZSLER and Head of IZSLER Parma.

November 2015
Q: Ten years ago, what technologies did you use for food pathogendetection and typing?
SP: Over the last decade, the diagnostic techniques we’ve used forpathogen detection have shifted from simple agar-based differential/
selective media and biochemical tests to PCR-based molecularmethods. The standard microbiology techniques are still usedwidely as initial diagnostics, but these methods don’t provide thedata necessary to perform pathogen typing for epidemiologicalinvestigations. They also don’t deliver the amount of informationneeded to depict the distribution of pathogens in the target population(animal or human) and along the food chain.

“With NGS, we can access all
the genetic variant informationthat a pathogen stores in its
chromosome as it’s spreadingin the environment from anintermediate host or foodto humans.”
Q: What made you consider adding NGS tools at your IZSLERParma laboratory?
SP: With NGS, we can access all the genetic variant informationthat a pathogen stores in its chromosome as it’s spreading in theenvironment from an intermediate host or food to humans. This typeof information is pivotal in supporting an epidemiological investigation.
We can also use NGS to identify the sources of infection and routes oftransmission, along with virulence and antimicrobial resistance genes.
The greatest advantage of NGS is that it enables us to use a single
method to obtain all this data, rather than performing multiple analyses
using several time-intensive techniques such as pulse-field gelelectrophoresis (PFGE), multiple-locus variable number tandem repeat
analysis (MLVA), multilocus sequence typing (MLST), or antibiograms.
Q: Why did you choose the MiSeq System over other NGS systems?
SP: We chose the MiSeq System because of its base calling quality,
the absence of homopolymer bias, and the high read length and yield
it can achieve at an affordable instrument cost.
Q: What types of sequencing do you perform with theMiSeq System?
SP: We have been using the MiSeq System to perform whole-genome
analyses of bacterial and viral isolates whenever a foodborne outbreakis suspected or detected within our regional surveillance mandate. The
speed and multiplexing capabilities of the MiSeq System enable us toperform epidemiology and genetic screening studies efficiently.
Q: What epidemiology studies have you performed with theMiSeq System?
SP: When we first got the MiSeq System, we wanted to implement
our pipelines and evaluate the potential of this new genomic tool.
So we used the MiSeq System to analyze retrospectively a humanoutbreak of infections by Salmonella enterica serovar Manhattan, aFor Research Use Only. Not for use in diagnostic procedures.

rare serovar in humans that occurred in Italy in 2009. In particular,
we performed a differential single nucleotide polymorphism (SNP)
analysis in comparison with the gold-standard genotyping method,
PFGE.2 In that study, a total of 39 isolates were analyzed frompatients, food, feed, animal, and environmental sources, resulting
in 5 different PFGE profiles. Isolates epidemiologically related tothe outbreak clustered within the same pulsotype, SXB_BS.0003,
without any further differentiation. Upon reanalysis by whole-genome
sequencing based on total core SNPs, 4 distinct groups of isolates
emerged within the outbreak pulsotype. Interestingly, outbreak-related
human isolates where distinct from food-origin isolates associatedwith the outbreak. Following detailed analysis based on different SNP
subsets, the same distinction was shown by nonsynonymous SNPs,
as well as SNPs at the second and first plus second codon positions.
Conversely, analyses derived from synonymous and third codon
position SNPs that are less sensitive to selective pressure, showedthat the food and human isolates clustered together. This indicatedthat all outbreak-related isolates constituted a single clone, which wasin line with the epidemiological evidence. This work proved to us that
subclonal genotyping based on differences in even a few SNPs (5–6)
is a powerful tool for our diagnostic routine. It also highlighted theimportance of carefully evaluating the output of bioinformatic analyses
and the data sets used. The study was an encouraging milestone,
taking advantage of the high fidelity of base calling, and therefore SNP
identification, of the MiSeq System.
Q: How long did it take to add the MiSeq System into your workflow?
SP: We adopted the MiSeq System in late October 2013 and included
it in our workflow (library preparation and running samples) within
1 month of installation. From a practical point of view, we were up
and running easily and obtained workable results for epidemiologicalapplications quickly. Preparation of sequencing libraries is the mostsensitive step of the process and it took some fine-tuning to obtainstable results. We found that it’s important to spend the time up frontin evaluating the quality of DNA input and setting up multiplexing
protocols to ensure precise and accurate data generation.

“We chose the MiSeq Systembecause of its base calling quality,
the absence of homopolymer
bias, and the high read lengthand yield it can achieve at anaffordable instrument cost.”
Q: How many sequencing runs do you perform each week?
SP: We usually perform 1–2, 2 x 250 paired-end runs per week. We
use the Nextera® XT Library Preparation Kit for creating sequencingready libraries of bacterial and viral DNA.
Sample number varies depending on the genome size of thepathogens. Considering a mean genome of 4.5 Mbp, we multiplex 10bacteria plus up to 30 viruses per run.

November 2015
Q: Why did you choose the Nextera XT Library Preparation Kit?
SP: We’re working with bacterial and viral clinical isolates and often
deal with extremely small samples. We chose the Nextera XT LibraryPrep Kit because it enables us to prepare high-quality libraries fromjust 1 ng of input DNA.

“We adopted the MiSeq System inlate October 2013 and included itin our workflow (library preparationand running samples) within 1month of installation.”
Q: What data analysis software do you use?
SP: Our data analysis pipelines are based almost exclusively on opensource software, which we use to perform sequencing reads QC, draft
genome assembly and annotation, variant detection, and phylogenetic
analysis of clinical isolates.
Q: Do you include sequencing data in the reports you provide
to customers?
SP: We are a public health laboratory, so our main NGS activities
are focused on epidemiological surveillance of human and animal
pathogens within the horizon of official surveillance plans. From thisperspective, we include the relevant details of our data analyses,
including sequencing data, in the technical reports sent to thecompetent authorities.

In addition, the MiSeq System can access all the information stored
in a pathogen’s genome, providing the data to infer correct pathogen
typing. In fact, the amount of informative genetic data the MiSeq
System provides is greater than the sum of the data generated using
all ‘traditional’ methods, enabling the identification of correct isolate
relatedness even without epidemiological metadata. With NGS costs
rapidly decreasing and data analysis pipelines being tested andvalidated worldwide, we look forward to replacing all our traditional
analytical methods with a single, dedicated NGS pipeline.

References1.

2.

Fitzgerald M, Thornton L, O’Gorman J, et al. Outbreak of a hepatitis A
infection associated with the consumption of frozen berries, Ireland, 2013—
linked to an international outbreak. Euro Surveill. 2014; 19(43) pii: 20942.
Scaltriti E, Sassera D, Comandatore F, et al. Differential single nucleotide
polymorphism-based analysis of an outbreak caused by Salmonella enterica
serovar Manhattan reveals epidemiological details missed by standard
pulsed-field gel electrophoresis. J Clin Microbiol. 2015; 53:1227–1238.

Learn more about the Illumina product and system
mentioned in this article:••“We chose the Nextera XT Library
Prep Kit because it enables us toprepare high-quality libraries fromjust 1 ng of input DNA.”
Q: How do you see NGS enhancing or expanding your service
offering in the future?
SP: Molecular epidemiology relies on precise typing of clinical andenvironmental isolates to solve important problems in public health,especially for outbreak investigations. The more variants, mainlySNPs, that we can describe for each single strain under scrutiny,
the higher the probability of finding the sources of infections androutes of transmission, along with virulence and fitness determinantsof pathogens.
In the past, this information was obtained using several different
analytical methods, each one having limitations and lengthy workflow
times that could jeopardize the fast response of health authorities toan infectious outbreak. The MiSeq System has proven it can provide
all this information from a single run quickly and efficiently. It takes just
3 days from clinical isolates in pure culture to analyzed sequence data.

For Research Use Only. Not for use in diagnostic procedures.

MiSeq System, www.illumina.com/systems/miseq.html
Nextera XT DNA Library Preparation Kit, www.illumina.com/products/nextera_xt_dna_library_prep_kit.html

November 2015

Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
For Research Use Only. Not for use in diagnostic procedures.
© 2015 Illumina, Inc. All rights reserved. Illumina, MiSeq, Nextera, and the pumpkin orange color are trademarks of Illumina, Inc. and/or itsaffiliate(s) in the U.S. and/or other countries. Pub. No. 770-2015-033 Current as of 02 November 2015