September 2015

A Tactical Approach To Eliminating Deadly Bacteria
Researchers are using the MiSeq® System to identify strains of drug-resistant bacteria fromaround the globe.
Introduction
Antimicrobial resistance is one of the global health care community’s
most pressing problems.1 When penicillin was first discovered in
1928, scientists hailed it as a miracle drug that turned life-threatening
infections into easily treatable conditions. By the 1940s, penicillin andother antibiotics were in wide use, saving thousands of lives worldwide.
As the decades passed, antibiotics were prescribed increasingly for
any condition resembling a bacterial infection. The overprescription in
man and agriculture, together with the misuse of leftover antibiotics,
changed the playing field in the fight against virulent bacteria.2
Antimicrobial resistant strains of Escherichia coli, Staphylococcus
aureus, and Clostridium difficile began to appear that even the mostpotent antibiotics were powerless against. According to the US
Centers for Disease Control and Prevention (CDC), at least 2 million
people become infected with drug-resistant bacteria every year in
the United States, with an estimated 23,000 people dying as a resultof those infections.3 It’s estimated that by 2050, antibiotic-resistantbacteria will kill a staggering 10 million people per year worldwide.4
This number, quite shockingly, will surpass the number of deaths
from cancer.5
One of the most dangerous antimicrobial resistant strains is methicillinresistant Staphylococcus aureus (MRSA) which first appeared in
1960.6 Initially, people were most likely to catch this drug-resistant“super bug” in hospitals. Today, new variants have spread in thegeneral community, increasing the likelihood of infection through abreak in the skin.
Henrik Westh, MD, Professor, PhD, a clinical microbiologist at
Hvidovre University Hospital in Copenhagen, Denmark, is using
next-generation sequencing (NGS) platforms, like the MiSeq System,
to understand the evolution and spread of MRSA and determine
the relatedness of different strains of MRSA acquired from aroundthe world. He and a team of global collaborators believe that byunderstanding how this deadly bacterium spreads, they can keep
MRSA at low levels within the population. They have used a techniquecalled “spa typing” which involves sequencing the MRSA spa geneto differentiate between various strains of the bacteria. iCommunity
spoke with Dr. Westh about his research and the global collaborationthat Dr. Westh hopes will quash drug-resistant super bugs, saving
millions of lives.
Q: What sparked your interest in clinical microbiology?
Henrik Westh (HW): I was a physician in clinical practice for 6 years
and the prospect of conducting microbiology research intrigued me.
It offered so many opportunities to make a difference for individualpatients and for improving infection control in hospitals. It’s exciting
to conduct research in antimicrobial resistance and contribute tosociety’s understanding about effective antimicrobial usage.

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

Q: What was sequencing technology like when you first began touse it in your studies?
HW: I began using Sanger sequencing performed with electrophoresis
gel glass plates. Sequencing genomes using next-generation
sequencing (NGS) technology really started around the time my firstPhD student was trying to analyze a staphylococcal chromosome
with long-range PCR. We couldn’t get PCR to work, so we decided tospend about $10,000 to have the genome sequenced on the Roche
454 system, the only sequencer at the time. It turned out that theregion she was looking at was not 10,000 base pairs long as we had
estimated, but a little over 35,000 base pairs. It would have taken hermonths to sequence it with Sanger, but NGS offered a much fastersolution. I thought, ‘wow, NGS works!’
Q: When did you start incorporating NGS into your studies routinely?
HW: We began using NGS in our studies in late 2009. At the time,
there were only a few places performing NGS and it was used mainly
for human genome sequencing. People believed that it was better
suited as a tool for research, or maybe in a reference or service
laboratory. Later, we realized it could be used for microbial genomes
and infection control.
Q: What impact have these NGS tools had on your research?
HW: NGS provides high-resolution data, enabling us to identify whichorganisms are part of the transmission chain and which aren’t in termsof infection control. We couldn’t do that with the old typing methods;
we couldn’t even see that we were making mistakes. NGS is a uniquetool that is almost mistake-free.

Dr. Henrik Westh is a clinical microbiologist at Hvidovre University
Hospital in Copenhagen, Denmark.

September 2015
Q: What is MRSA?
HW: MRSA is an S. aureus bacteria that has become resistant tobeta-lactam antibiotics. This includes members of the penicillin andcephalosporin antibiotic groups, which are the first and best choiceantibiotics for treating seriously ill patients. MRSA was made by man
using and misusing antibiotics. The agricultural industry has createdlivestock-associated MRSA as well, by using massive amounts ofantibiotics especially in pig production.
MRSA has evolved quite a lot in the past 15 years. In the 1980s and1990s, you only acquired it in hospitals. In the late 1990s, it became
prevalent in the community. There are 3 main MRSA variants in the
population today; hospital clones, community clones, and livestockassociated clones.

“NGS provides high-resolution
data, enabling us to identify
which organisms are part of thetransmission chain and which
aren’t in terms of infection control.”
Q: Why is typing MRSA strains important?
HW: Typing MRSA strains is important for infection control. If we have
2 cases of MRSA in the hospital, we want to know if they are related.
If the strains are identified in the same hospital room, we know thatthey are connected. But if they are isolated from different departments,
it might be the result of a transmission chain that we need toidentify immediately.
Q: When did you begin focusing on MRSA?
HW: I began studying MRSA while writing my clinical microbiology
thesis on S. aureus. At first, I was working on issues related toordinary S. aureus, its antimicrobial resistance, and its spread in
hospitals. We began looking at MRSA’s evolution studying a large
outbreak in Denmark in the 1960s, which followed an outbreak 2years earlier in the UK and other parts of Europe. We found that about
20% of all bacteremic patients with S. aureus in Denmark had MRSA.
And it was from a single clone.
We looked at how the epidemic evolved and the factors allowing us
to control it. We saw, by pulsed-field gel electrophoresis (PFGE), thatthe old Danish MRSA isolates looked like some of the more modern
clones from a Portuguese hospital. This led us to introduce spa typing.
Q: What is spa typing?
HW: spa (staphylococcus protein A) is a gene in the S. aureus
chromosome. spa typing is a sequenced-based, universal tool. It has
been very valuable in determining whether patients have transmitted
their bacteria between each other. When I started, there wereprobably less than 200 known variants. Using spa typing, the scientificcommunity has identified 15,000 variants to date. Researchers can
now access the SpaServer (Seqnet.org), a fantastic international
database that was created by the initiatives of Drs. Dag Harmsen
and Alexander Friedrich in Germany and today contains more than
330,000 isolates.7

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

When I became a physician, there were fewer than 100 patients a year
in Denmark that had MRSA. In 2003, I could see this curve changing,so we introduced spa typing for all our MRSA isolates. We suggested
that the National Health Board of Denmark revise the country-wide
MRSA guidelines, and ended up with new national guidelines in 2006.
We have many rules about how to ‘search and destroy’ MRSA. We’retrying to make sure it doesn’t spread in the community, or take up
residence in our hospitals and thereby infect patients.
Q: How is the spa gene used to type MRSA strains?
HW: spa typing is the most reproducible and user-friendly typing
method. The spa sequence has several repeats, usually spanningabout 24 base pairs each, that are repeated between 1–25 times
within the gene. Each repeat can be different and there are many(+680) distinct types of repeats. Every time a unique sequenceis identified, the spa type is given a number and is added to theSpaServer database.
Q: How did the spa typing data from the MiSeq System and Sanger
sequencing compare?
HW: With the introduction of routine WGS of MRSA, we needed toensure backward compatibility with our old sequence-based typing
method. In 2014, we published data that compared spa typing using
the MiSeq System and Sanger sequencing.8 There was over 97% total
agreement between the data sets. We’re so satisfied with the MiSeq
System data quality that we don’t perform spa typing with Sanger
sequencing anymore.
The speed of the 2 methods is about the same, but instead of justgetting the spa type, the MiSeq System delivers data on the wholegenome with the same turnaround time. That gives us more datafor infection control and for follow-on research projects. In particular,
single nucleotide polymorphism (SNP) tree analysis has shown us that
SNP trees give resolution even within a spa type.9
We now test 24 genomes in a single run and run the MiSeq System
3 times a week. As Illumina NGS technology continues to improve,
we’re looking forward to even faster turnaround time and the ability tosupport automated library prep.

“We’re so satisfied with the MiSeq
System data quality that we don’t
perform spa typing with Sanger
sequencing anymore.”
Q: Is whole-genome sequencing (WGS) performed on all MRSA
isolates at Hvidovre Hospital?
HW: In 2012, we began performing WGS on the MiSeq System forspa typing of all MRSA isolates at the hospital. We’re also the regionalreference laboratory, performing spa typing for all hospitals in thecapital region.
In the beginning, I’m sure microbiologists in Denmark were thinking,‘Why is he doing this? Why is he sequencing all MRSA isolates?’ I’m
now seeing more microbiologists performing WGS and we alreadyhave one more microbiology department in another Danish hospitalSeptember 2015that has bought a MiSeq System. In the next year, we intend to obtain
more MiSeq Systems so that we can perform genome sequencing
in all 3 facilities in our hospital network. We’re also making sure that
all the data ends up in a common hospital database so that we canshare our information.
Q: What type of database do the spa typing results end up in?
HW: Results from spa typing, multilocus sequence typing (MLST), andPanton-Valentine leukocidin (PVL) gene presence go directly into ourlaboratory information system and the patient’s records. This way
clinicians and microbiologists know exactly what types of clones arepresent. With the SNP-based analysis systems that we have now, wecan see that isolates we thought were identical, were very different
actually. We are creating a national database built on NGS dataand patient epigenetics data. International databases will become
very important as we can share sequences and develop common
nomenclature for identification and naming of clones.

“...instead of just getting the spa
type, the MiSeq System delivers
data on the whole genome withthe same turnaround time.”
Q: Have you used the MiSeq System for any other microbial
sequencing projects?
HW: To date, we’ve sequenced 4500 bacterial genomes. We’ve
sequenced Vancomycin-resistant enterococci,10 and several E. coli,
especially looking for the plasmids that are responsible for beta-lactam
resistance and about 10 other species. We are collaborating withcolleagues at the Technical University of Copenhagen to perform
metagenomic analysis. For example, we performed a study looking at
the fecal microbiome to identify causes of infectious diarrhea. Routine
metagenomic analysis of selected patient samples and study of viral
populations will begin in 2016.
Q: When you became a clinical microbiologist, did you believe that
there would be a day when WGS could be performed in 1 day?
HW: I became a microbiologist in the late 1980s and at the time it was
fantastic when somebody would complete a PhD on the sequence ofa plasmid, phage, or a transposon. Thinking back it’s really incredible
the technological strides that we’ve made.

For example, we have an influenza PCR assay, but the influenza
virus is a moving target. During the H1N1 epidemic several years
ago, none of the PCR assays in the world were perfect. So we must
make sure that our molecular assays are working perfectly every year.
I think NGS will improve the quality and quality control of all thesemolecular methods.

“There’s a lot of evolutionary
history of microbial-resistantstrains that we can trackwith NGS.”
Q: How will the declining cost of sequencing benefit clinicalmicrobiology research?
HW: Low-cost sequencing will benefit research and infection control
efforts. In Germany, if there is an outbreak in a hospital ward, theauthorities close the ward and the hospital loses money until theoutbreak is controlled. With NGS, you can look at suspected outbreakisolates, identify the infection points, and reopen the ward faster. NGS
is also fantastic for identifying the source of food outbreaks. Last
year we had a listeria outbreak from contaminated sausages. WGS
identified that all of the isolates were identical and from the samefactory. The outbreak was halted by shutting down the factory.
Dr. Peter Gerner-Smidt from the CDC has said that the preferred
method PFGE is in the process of being replaced by NGS for
PulseNet, which is used globally for the surveillance of foodborne
diseases. This has worked very well, but it will become even easier
to share information with NGS. Like PFGE, it’s universal, electronic,portable and definitive.
Q: What are the next steps in your MRSA research?
HW: We are analyzing various MRSA, using our NGS and SNP trees
to understand its epidemiology. MRSA is brought into Denmarkby Danish people traveling around the world, and now we can say
whether those isolates are identical or whether MRSA acquired in
the Near East is different from MRSA acquired in India. There’s a lotof evolutionary history of microbial-resistant strains that we can trackwith NGS. It’s quite interesting.

Q: How has NGS transformed clinical microbiology?
HW: We have already talked about its benefits in infection controland typing. We are heading towards a better understanding ofmultimicroorganism infections and suitable metagenomics approaches
will improve diagnosis and treatment. All the sequence data wegenerate will help make better PCR-based assays for faster screening.

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

September 2015

References1.

WHO’s first global report on antibiotic resistance reveals serious, worldwide
threat to public health. Geneva, Switzerland; World Health Organization;
April 30, 2014. www.who.int/mediacentre/news/releases/2014/amr-report/
en/. Accessed July 16, 2015.
2. US Centers for Disease Control and Prevention. About AntimicrobialResistance. CDC web page. www.cdc.gov/drugresistance/about.html.
Accessed July 16, 2015.
3. US Centers for Disease Control and Prevention. Antibiotic ResistanceThreats in the United States, 2013, www.cdc.gov/drugresistance/pdf/arthreats-2013-508.pdf. Accessed July 16, 2015.
4. Review on antimicrobial resistance. May 14, 2015. amr-review.org/.
Accessed July 25, 2015.
5. Overcoming Cancer in the 21st Century, UCL School of Pharmacy, July 14,
2015, www.ucl.ac.uk/pharmacy/departments/practice-policy/VF_Embargo.
pdf. Accessed July 16, 2015.
6. Boucher HW and Corey GR. Epidemiology of Methicillin-Resistant
Staphylococcus aureus. Clin Infect Dis. 2008; 1:46 Supple 5:344–349.
7. Ridom Spa Server, www.spaserver.ridom.de/. Accessed July 16, 2015.
8. Bartels MD, Petersen A, Worning P, et al. Comparing Whole-Genome
Sequencing with Sanger Sequencing for spa Typing of Meticillin-Resistant
Staphylococcus aureus. J Clin Microbiol. 2014; 52(12): 4305–4308.
9. Bartels MD, Larner-Svensson H, Meiniche H, et al. Monitoring meticillin
resistant Staphylococcus aureus and its spread in Copenhagen, Denmark,2013, through routine whole-genome sequencing. EuroSurveill. 2015;20(17). pii: 21112.
10. Pinholt M, Larner-Svensson H, Littauer P, et al. Multiple hospital outbreaksof vanA Enterococcus faecium in Denmark, 2012-13, investigated by WGS,MLST and PFGE. J Antimicrob Chemother. 2015; pii: dkv142. [Epub aheadof print].

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© 2015 Illumina, Inc. All rights reserved. Illumina, MiSeq, and the pumpkin orange color, and the streaming bases design are trademarksof Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. Pub. No. 1270-2015-003 Current as of 01 September 2015