09Apr / 2018

Responding to Emerging and Zoonotic Infectious Disease Threats in 2017

Montage of photos. From left: a photo of different raw foods, including salmon, fruits and vegetables. A photo of a boy taking an oral vaccine. A photo of bacteria growing in petri dish.

Photo of Rima F. Khabbaz, MD, Director, National Center for Emerging and Zoonotic Infectious Diseases — Rima F. Khabbaz, MD, Director, National Center for Emerging and Zoonotic Infectious Diseases

The fungal superbug Candida auris causes serious and often fatal infections. It can strike people in the places where they seek care—hospitals and other healthcare facilities. In early 2016, we knew about outbreaks of C. auris infections on multiple continents, but we were not sure whether C. auris was in the United States. Fast forward to 2017: C. auris is a priority for public health workers in the United States, and CDC, along with state and local health departments, has tracked more than 200 cases of C. auris infection in the country. Our experts have worked with healthcare facilities across the nation to implement infection control measures and stop transmission.

The progress to track and prevent C. auris is just one example of the important work experts from CDC’s National Center for Emerging and Zoonotic Infectious Diseases (NCEZID) tackled in 2017. Some of the other highlights from the NCEZID 2017 Accomplishments report are described below.

A tremendous year for public health

Summarizing last year’s major efforts was a difficult task. The numbers alone depict a tremendous year for public health. Here are just a few examples. CDC sequenced nearly 45,000 DNA samples by using Advanced Molecular Detection (AMD) technologies. The agency identified more than 1,100 illnesses that were associated with backyard flocks—the highest number ever recorded by CDC in a single year. And the Antibiotic Resistance Lab Network performed more than 12,000 tests to contain the spread of resistant infections, just to name a few accomplishments.

Tracking new and evolving threats

Every day we are learning more about antibiotic resistance, which continues to be among the biggest health concerns in our country. In 2017, CDC took several important steps to combat antibiotic resistance, including rolling out a containment strategy to slow the spread of drug-resistant diseases in healthcare facilities—starting with a single case—and supporting 25 innovators through a CDC pilot project to develop solutions to antibiotic resistance crises.

Understanding the impact

We are also learning more about Zika virus. Zika was often in the headlines in 2016 and 2017, and the mosquito-borne virus continues to be a threat, especially for pregnant women and their fetuses. Last year, CDC experts shed light on a lesser-known effect of Zika virus infection: a link with Guillain-Barré syndrome (GBS), an uncommon illness of the nervous system. In 2017, CDC and partners conducted the first case-control study in the Americas that showed evidence linking Zika virus infection and GBS. This was just one of many vector-borne diseases CDC tackled in 2017.

Responding to new outbreaks

As we continued to work on lingering threats like antibiotic resistance and Zika, CDC also responded to new outbreaks in 2017, both at home and abroad. In the United States, we saw a range of illnesses connected to food products—from Salmonella infections linked to papayas to an Escherichia coli outbreak from soy nut butter. For the first time, scientists linked an outbreak of Seoul virus infections to pet rats in the United States, and AMD lab techniques proved critical in tracing this and other outbreaks. CDC scientists traveled across the globe in 2017 to investigate a myriad of outbreaks, including an outbreak of anthrax infections in animals in Namibia that posed a threat to human health. Experts helped respond to yellow fever outbreaks in countries including Brazil, and we continue that work today as the yellow fever outbreak in Brazil has expanded over the past two years and could affect US travelers.

Like CDC’s response to yellow fever outbreaks, much of last year’s work continues in 2018. We are closely tracking emerging infections like C. auris, continuing to study the effects of unusual diseases like Zika, and investigating and containing outbreaks of infections caused by a wide range of microbes such as Salmonella bacteria, monkeypox virus, and hemorrhagic fever viruses.

Want to learn more? Read the full NCEZID 2017 Accomplishments report, and follow NCEZID on Twitter @CDC_NCEZID.

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30Jan / 2018

Engineered bacteria to detect gut inflammation

0000-0002-8715-28960000-0003-0319-5416 I wrote about engineered probiotics at the beginning of 2017, and the field continued throughout 2017 with more papers and startup news using engineered bacteria in the gut. For instance, one paper used engineered

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08Feb / 2017

PulseNet key to solving 2010 E. coli outbreak linked to lettuce

by Kim Krisberg

On April 22, 2010, federal public health officials notified the New York State Department of Health of two E. coli clusters at colleges in Michigan and Ohio. The very next day, the New York agency got word of an illness cluster in its own state with symptoms similar to the neighboring outbreaks.

Fortunately, that initial notification came via PulseNet, the national molecular subtyping network for foodborne disease surveillance, which allows public health scientists and investigators to rapidly identify foodborne illness outbreaks. That meant staff at the New York State public health laboratory, officially known as the Wadsworth Center, had easy access to Michigan’s and Ohio’s laboratory findings, which allowed immediate testing to begin to discover whether the New York illnesses were connected to the larger outbreak. Just a handful of days later, the New York lab had an answer — DNA fingerprints from patient specimens in Michigan, Ohio and New York were a match. The E. coli O145 outbreak had spread to New York.

“It was invaluable for us,” said Madhu Anand, DrPH, deputy director of the Regional Epidemiology and Investigations Program in the department’s Bureau of Communicable Disease Control, of PulseNet, which celebrated its 20th anniversary last year. “PulseNet was critical at every stage of this investigation.”

Just a few days following identification of the initial New York illness cluster, which occurred at a college in western New York, public health staff got word about a cluster of hemolytic uremic syndrome (HUS) illnesses in a school district just north of New York City. HUS is a potentially life-threatening complication associated with Shiga toxin-producing E. coli infection. Public health workers began active surveillance in the district, Anand said, finding multiple cases that matched the profile of cases connected to the E. coli outbreak.

Around this same time, CDC announced that epidemiologic and traceback investigations in Michigan and Ohio pointed to shredded romaine lettuce from a single distributor as the culprit. In response, the New York State Department of Health worked with local public health to collect any leftover lettuce from the college. The college didn’t have any leftovers, said David Nicholas, MPH, research scientist and epidemiologist in the state’s Bureau of Community Environmental Health and Food Protection, but it did have an invoice, which showed the same distributor identified in Ohio and Michigan. Public health staff also sought out lettuce leftovers in the affected school district, and they found plenty.

On April 28, 2010, the Wadsworth Center received more than 150 pounds of shredded lettuce from the school district — or what Nicholas described as a “Honda full of lettuce.” Lab staff got to work testing portions of the entire lot, which were divided into two-pound bags, reported Nellie Dumas, associate director of the Wadsworth Center’s Bacteriology Laboratory. However, one of the two-pound bags was stamped with an expiration date indicating it could have been among the same batch of shredded lettuce that the sickened children had eaten. That expiration date led lab staff to test the entire two pounds of lettuce, Dumas said.

In testing that particular bag of lettuce, laboratorians were able to isolate E. coli O145, which was then tested by pulsed-field gel electrophoresis (PFGE) to obtain a DNA fingerprint. The DNA fingerprint matched the outbreak strains identified in Ohio and Michigan. The Wadsworth findings were then uploaded to PulseNet, helping to confirm that shredded lettuce was indeed the source of the outbreak, said Deborah Baker, research scientist in the Wadsworth Center Bacteriology Laboratory.

“PulseNet was vitally important because it allowed states to instantly share subtyping information,” Baker said. “As soon as we have a PFGE pattern, we can immediately go into the database and see what’s happening in other states.”

Overall, according to Anand, New York state was home to six confirmed cases and one probable case of E. coli O145 connected to multistate outbreak traced back to shredded lettuce. All six confirmed patients had to be hospitalized and four developed HUS. Nationwide, according to CDC, 26 confirmed and seven probable cases of illness were connected to the E. coli outbreak in five states: Michigan, New York, Ohio, Tennessee and Pennsylvania. (The cases in Tennessee and Pennsylvania were identified in retrospect using PulseNet data.) Among the 30 E. coli patients with available information, 40 percent became so sick they had to be hospitalized. Thankfully, no deaths occurred.

A May 10, 2010 news release from the U.S Food and Drug Administration linked the contaminated shredded lettuce back to Freshway Foods in Ohio. The company issued a voluntary recall.

“For 20 years, PulseNet has helped us find the sources of these horrific illnesses,” said Dumas, associate director of the Wadsworth Center Bacteriology Laboratory. “It’s total teamwork.”

According to CDC, PulseNet identifies about 1,500 clusters of foodborne illness every year, about 250 clusters that cross state lines, and about 30 multistate outbreaks traced back to a food source. A recent economic evaluation of PulseNet found that every year, the laboratory network prevents more than 266,500 illnesses from Salmonella, nearly 9,500 illnesses from E. coli and 56 from Listeria. That translates into $507 million in reduced medical and productivity costs.

The post PulseNet key to solving 2010 E. coli outbreak linked to lettuce appeared first on APHL Lab Blog.

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08Dec / 2016

With PulseNet, handful of E. coli cases reveal multistate outbreak, prompt huge recall

By Kim Krisberg

In 2014, two Ohio residents living more than 100 miles apart were diagnosed with an E. coli infection. Twenty years ago, the two cases might have been chalked up to coincidence — after all, tens of millions of Americans experience foodborne illness every year.

But thanks to a nationwide lab network known as PulseNet, public health officials could compare the genetic patterns of the Ohio cases to foodborne illness cases across the country, eventually detecting a multistate foodborne illness outbreak that led to the recall of 1.8 million pounds of ground beef products. Overall, 12 people across Ohio, Michigan, Massachusetts and Missouri were diagnosed with outbreak strains of Shiga toxin-producing Escherichia coli O157:H7, or STEC O157:H7, and more than half of those sickened had to be hospitalized.

“Without PulseNet, we may have never recognized this as a multistate outbreak,” said Scott Nowicki, MPH, epidemiologist at the Ohio Department of Health.

Several key activities came together in late spring 2014 that enabled public health officials in Ohio and Michigan to detect and contain the outbreak fairly quickly. First, after the Ohio Public Health Laboratory confirmed the initial two cases of STEC O157:H7, student interviewers with Ohio’s FoodCORE team — a Centers for Disease Control and Prevention-funded effort to strengthen state and local foodborne illness outbreak response — set out to interview the patients. It turned out both patients, who lived more than 100 miles apart, said they had eaten at the same local chain restaurant that specializes in serving undercooked hamburgers. It was a strong signal that undercooked beef, as opposed to contaminated produce, was the culprit, Nowicki said.

On the same day as the FoodCORE interviews, the Ohio Public Health Laboratory uploaded its test results for the local STEC O157:H7 cases to PulseNet. Previously, the lab, which routinely receives specimens of public health importance from health providers around the state, received isolates connected to the STEC O157:H7 patients. Lab staff then performed pulsed-field gel electrophoresis, or PFGE, to determine the sample’s DNA fingerprint pattern. They posted the fingerprint patterns to PulseNet and quickly noticed their PFGE results matched two isolates in Michigan.

With PulseNet, handful of E. coli cases reveal multistate outbreak, prompt huge recall | www.APHLblog.org In addition to a match, the PFGE pattern was also relatively uncommon, which was another strong signal of an outbreak rather than a string of isolated cases, said Eric Brandt, a laboratory scientist at the Ohio Department of Health, Bureau of Public Health Laboratory.

With the matching PFGE results, epidemiologists in Ohio and Michigan began comparing notes, finding that patients in both states reported eating at restaurants that serve undercooked beef. In particular, 92% of the 12 ill persons identified in the outbreak reported eating ground beef at such a restaurant before they became sick, and 73% said they may have eaten hamburger prepared rare, medium rare or undercooked.

From there, an intensive local, state and federal traceback investigation ensued, eventually tracing the ground beef at the restaurants where the STEC O157:H7 patients had eaten to the Wolverine Packing Company in Detroit. In May 2014, the meatpacking company recalled about 1.8 million pounds of ground beef that may have been contaminated with the pathogen. During the outbreak, five people were sickened in Ohio, five in Michigan, one in Massachusetts and one in Missouri. While seven of those people had to be hospitalized, none developed hemolytic uremic syndrome, a potentially life-threatening complication associated with STEC O157:H7 infection.

Nowicki noted that before PulseNet, it often took many more cases of foodborne illness for public health officials to recognize an outbreak and begin efforts to identify the source and prevent further disease. Indeed, he said the 2014 STEC O157:H7 outbreak is the perfect example of how PulseNet can quickly connect a small handful of seemingly isolated dots to reveal the outbreak lurking beneath.

“For identifying outbreaks,” Nowicki said, “PulseNet is invaluable.”

Brandt agreed, adding that “these very sporadic cases that cross state lines…those would have been much more difficult to detect in the pre-PulseNet days.” He also said that PulseNet, which celebrated its 20th anniversary this year, is “fundamental” to the Ohio Public Health Laboratory’s foodborne illness capacity, providing the lab’s primary infrastructure for cluster detection, bacterial subtyping, training, instrumentation and much more.

“PulseNet is crucial,” said Brandt, who’s spent most of his career working with PulseNet. “I can’t even imagine what it was like before.”

The post With PulseNet, handful of E. coli cases reveal multistate outbreak, prompt huge recall appeared first on APHL Lab Blog.

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29Jun / 2016

Sprouts: Just say no?

By Caitlin Saucier Feltner, BSN, RN

Most people have seen or eaten sprouts, the crunchy mild-tasting greens that are often incorporated into stir-fried dishes and nestled into sandwiches to add texture and color. Did you know that many food safety experts list sprouts at the top of their “Do Not Eat” lists? The term “sprouts” actually refers to a variety of small plants that can be sprouted from several different seeds including alfalfa, broccoli, mung beans and radish. While they can be an excellent source of antioxidants, vitamins and minerals, sprouts are also frequently contaminated with foodborne pathogens, some of which can be quite dangerous. What makes sprouts so risky? The answer lies in how they are grown, processed and stored before they get to your plate.

While there is nutritional value in sprouts, there can also be significant risk. Raw sprouts can be a carrier for harmful bacteria including Salmonella, Listeria and E. coli. Some of these pathogens can cause more than just an upset tummy. For example, Shiga toxin-producing E. coli (STEC) are particularly dangerous. Of those infected with STEC, 5-10% develop hemolytic uremic syndrome (HUS) which can result in permanent kidney damage and even death. Listeria has a mortality rate of about 21%,and carries a particularly high risk for pregnant women and those with compromised immune systems. Consumption of raw sprouts can have health consequences that are more than theoretical and can be extremely serious.

Since 1996, there have been more than 30 outbreaks linked to sprouts that have sickened at least 1,800 people. (There have probably been far more cases than this because not every illness is confirmed and reported.) Perhaps the most well-known of the sprout related outbreaks, Jimmy John’s Gourmet Sandwiches served sprouts eventually linked to outbreaks in 2008, 2009, 2010 (two outbreaks), 2012 and 2014. While the company temporarily removed sprouts from their menu, sprouts have returned in many states and some customers placing online orders have been met with a pop-up safety warning.

Sprouts: Just say no? | www.APHLblog.org So what is it about sprouts that makes them so risky? According to the Colorado Integrated Food Safety Center for Excellence, the source of bacterial contamination for sprouts is nearly always the seeds.

The plants that initially produce these seeds are grown in typical agricultural settings. Animal manure, contaminated water and poor worker hygiene are all potential ways for the seeds to come in contact with harmful bacteria. The seeds can also become contaminated while being harvested, during storage or transportation (for example, by rodents and pests that live in a storage facility). Additionally, seeds intended to be sprouted sometimes undergo a process known as scarification to break down the seed’s outer coat and let in water. This process speeds up sprouting, but also leaves tiny holes that trap bacteria.

Some of these contaminated seeds are then sent to a sprouting facility where they are exposed to warm and moist conditions to encourage their growth. Unfortunately, these conditions are also perfect for bacterial growth. In this ideal environment, bacteria undergo what is known as “exponential growth.” The concept is fairly easy to understand – one bacterium doubles into two which then doubles into four and so forth. Just a few bacteria attached to a seed can quickly become a critical mass. This bacteria then grows from the seed into the sprout itself.

There is also opportunity for the sprout itself to become contaminated if it is exposed to poor sanitation conditions such as improperly cleaned equipment. While some sprouting facilities use a chemical soak on the seeds to reduce the number of bacteria, less than half of sprouting operations use a disinfection treatment before sprouting since the final sprout output may be reduced.

There is a common misconception that sprouts grown outside of a commercial environment, such as in an individual’s home garden, are safe from the risk of contamination. That’s simply untrue. Because the source of bacterial contamination is almost always the seed, a home gardener’s attention to good hand hygiene and a clean growing environment does not exempt them from the risk associated with sprouts. The contaminated seeds mentioned above are likely the same ones that a home gardener would find in a seed packet. That means home sprouters are equally likely to be starting the process with harmful bacteria present.

The inevitable next question asked by sprout-lovers: can’t I just wash them? Because contamination almost always grows from the seed into the plant, washing sprouts won’t remove those risky pathogens. Per FDA recommendations, the safest practice is to cook sprouts thoroughly. I realize this isn’t always the best way to serve sprouts, but it is truly the most effective way to ensure foodborne pathogens are eliminated. This is even more critically important when they will be served to children, older adults, pregnant women and those with poor immune system function.

Like with anything, most food safety experts would say that they always weigh the risks of a particular food with the benefits. Is your love of sprouts great enough to warrant the risk? That’s for you to decide. While eating raw sprouts will certainly not lead to illness every time, the high risk of contracting a dangerous pathogen is enough of a deterrent for me. Dine safely!

Caitlin Saucier Feltner is a former CDC/APHL Emerging Infectious Diseases Laboratory Training Fellow who worked with the Hawaii Department of Health State Laboratories Division. Read more of Caitlin’s posts.

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19Oct / 2015

Lessons learned as a food safety professional

We asked some of our food safety program staff and committee members a simple question: How has your work changed the way you eat and/or think about food? Here are their responses…

Lessons learned as a food safety professional | www.APHLblog.org Before I start prepping any high-risk food like raw meat, I make sure the area is clear of anything I might not want to contaminate (especially if you are running water which can create aersolization). I put clean dishes from a drying rack away, and move other utensils that may normally be on the counter (like a utensil jar) completely out of the way of the food preparation process. Why? Here is one example… There was a small Salmonella outbreak that began with an infant getting salmonellosis. Upon investigation it was determined that the family cleaned out the turtle aquarium in the kitchen sink near where the baby bottles had been left out to dry; the aerosolization of that process contaminated the bottles which then passed the organism to the infant. While it makes perfect sense, it is not something I would have considered prior to my work in food safety.
– Stephen Gladbach
Microbiology Unit Chief
Missouri State Public Health Laboratory

I am much more aware of the connection between agriculture practices and the safety of the food I prepare for my family. The way cows and chickens are raised is not only important for animal wellbeing. On-farm practices impact the number and types of pathogens that may end up in our food, in our kitchens and on our plates. Food safety is not as simple as “wash your hands” and “wash your produce.” Those are very important steps we should take to protect ourselves, but safety begins on the farm.
– Shari Shea
Director, Food Safety
APHL

The one thing I have learned is to be diligent in good food preparation practices at home to reduce risk to my family. This includes using different cutting boards and knives for the foods I am preparing; to wash my vegetables more than a quick rinse under the faucet; to make sure meat is cooked to the proper temperature; and good clean-up afterward. When eating out, I look around to see the cleanliness of the place. If the room that many customers see (bathroom) is dirty, what does that say about places I can’t see?
– George Goedesky
Vice President, MALDI Biotyper
Bruker Daltonics Inc.

I’ve heard too many stories about children who were gravely ill or even died from being infected with E. coli O157:H7, a dangerous bacteria often associated with undercooked ground beef. I’m hesitant to serve my kids hamburgers, but when I do I make sure they’re cooked to a minimum temperature of 160°F. A good food thermometer is one of the most important kitchen utensils to have on hand. Everyone should own one.
– Kirsten Larson
Manager, Food Safety
APHL

Read more posts about food safety:

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08Oct / 2015

Wrap up of talk by Rich Lenski at UC Davis

Rich Lenski gave a talk today at UC Davis - part of a two talk series. This was a presentation more for the public and tomorrow he gives one more for the science crowd. Today's talk was a really nice overview of Lenski's work on long term evolution experiments in E. coli. I made a Storify of the tweets about the talk:

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30Jan / 2015

SynBio B***********: Genetic recoding. Also, measles goes to Disneyland

An early triumph for the infant synthetic biology? Do you suppose Science‘s Breakthrough (Arrrrgh!) of the Year for 2015 has already arrived? In January, no less? Via two papers in Nature? Which venue, I suppose, might take it out … Continue reading »

The post SynBio B***********: Genetic recoding. Also, measles goes to Disneyland appeared first on PLOS Blogs Network.

13Apr / 2013

Bacterial genomics tutorial

This is a shameless plug for an article and accompanying tutorial I’ve just published together with David Edwards, my excellent MSc Bioinformatics student from the University of Melbourne. It’s currently available as a PDF pre-pub from BMC Microbial Informatics and Experimentation, but the web version will be available soon. The accompanying tutorial is available here.

The idea for this came from discussions at last year’s ASM (Australian Society of Microbiology) meeting, where it was highlighted that there was a lack of courses and tutorials available for biologists to learn the basics of genomic analysis so that they can make use of next gen sequencing. Michael Wise, a founding editor of BMC Microbial Informatics and Experimentation based at UWA in Perth, suggested the new journal would be an ideal home for such a tutorial… so here we are:

Beginner’s guide to comparative bacterial genome analysis using next-generation sequence data

http://www.microbialinformaticsj.com/content/3/1/2/

High throughput sequencing is now fast and cheap enough to be considered part of the toolbox for investigating bacteria, and there are thousands of bacterial genome sequences available for comparison in the public domain. Bacterial genome analysis is increasingly being performed by diverse groups in research, clinical and public health labs alike, who are interested in a wide array of topics related to bacterial genetics and evolution. Examples include outbreak analysis and the study of pathogenicity and antimicrobial resistance. In this beginner’s guide, we aim to provide an entry point for individuals with a biology background who want to perform their own bioinformatics analysis of bacterial genome data, to enable them to answer their own research questions. We assume readers will be familiar with genetics and the basic nature of sequence data, but do not assume any computer programming skills. The main topics covered are assembly, ordering of contigs, annotation, genome comparison and extracting common typing information. Each section includes worked examples using publicly available E. coli data and free software tools, all which can be performed on a desktop computer.

Four great tools

In the paper and tutorial, we introduce the four tools which we rely on most for basic analysis of bacterial genome assemblies: Velvet, ACT, Mauve and BRIG. All except ACT were developed as part of a PhD project, and have endured well beyond the original PhD to become well-known bioinformatics tools. New students take note!

In the paper, each tool is highlighted in its own figure, which includes some basic instructions. This is reproduced below, but is covered in much more detail in the tutorial that comes with the paper (link at the bottom).

1. Velvet for genome assembly

Possibly the most popular and widely used short read assembler, developed by the amazing Dan Zerbino during his PhD at EBI in Cambridge. Quite a PhD project!

[ Download | Paper | Protocol ]

Reads are assembled into contigs using Velvet and VelvetOptimiser in two steps, (1) velveth converts reads to k-mers using a hash table, and (2) velvetg assembles overlapping k-mers into contigs via a de Bruijn graph. VelvetOptimiser can be used to automate the optimisation of parameters for velveth and velvetg and generate an optimal assembly. To generate an assembly of E. coli O104:H4 using the command-line tool Velvet:

• Download Velvet [23] (we used version 1.2.08 on Mac OS X, compiled with a maximum k-mer length of 101 bp)

• Download the paired-end Illumina reads for E. coli O104:H4 strain TY-2482 (ENA accession SRR292770)

• Convert the reads to k-mers using this command:

velveth out_data_35 35 -fastq.gz -shortPaired -separate SRR292770_1.fastq.gz SRR292770_2.fastq.gz

• Then, assemble overlapping k-mers into contigs using this command:

velvetg out_data_35 -clean yes -exp_cov 21 -cov_cutoff 2.81 -min_contig_lgth 200

This will produce a set of contigs in multifasta format for further analysis. See Additional file 1: Tutorial for further details, including help with downloading reads and using VelvetOptimiser.

2. ACT for pairwise genome comparison

Part of the Sanger Institute’s Artemis suite of tools. Also look at Artemis (single genome viewer), DNA Plotter (which can draw circular diagrams of your genomes) and BAMView (which can display mapped reads overlaid on a reference genome), they are all available here.

[ Download | Paper | Manual ]

Artemis and ACT are free, interactive genome browsers (we used ACT 11.0.0 on Mac OS X).

• Open the assembled E. coli O104:H4 contigs in Artemis and write out a single, concatenated sequence using File -> Write -> All Bases -> FASTA Format.

• Generate a comparison file between the concatenated contigs and 2 alternative reference genomes using the website WebACT.

• Launch ACT and load in the reference sequences, contigs and comparison files, to get a 3-way comparison like the one shown here.

Here, the E. coli O104:H4 contigs are in the middle row, the enteroaggregative E. coli strain Ec55989 is on top and the enterohaemorrhagic E. coli strain EDL933 is below. Details of the comparison can be viewed by zooming in, to the level of genes or DNA bases.

3. Mauve for contig ordering and multiple genome comparison

Developed by the wonderful Aaron Darling during his PhD, he is now Associate Professor at University of Technology Sydney. Also see Mauve Assembly Metrics, an optional plugin for assessing assembly quality which was developed for the Assemblathon.

[ Download | Paper | User Guide ]

Mauve is a free alignment tool with an interactive browser for visualising results (we used Mauve 2.3.1 on Mac OS X).

• Launch Mauve and select File -> Align with progressiveMauve

• Click ‘Add Sequence…’ to add your genome assembly (e.g. annotated E. coli O104:H4 contigs) and other reference genomes for comparison.

• Specify a file for output, then click ‘Align…’

• When the alignment is finished, a visualization of the genome blocks and their homology will be displayed, as shown here. E. coli O104:H4 is on the top, red lines indicate contig boundaries within the assembly. Sequences outside coloured blocks do not have homologs in the other genomes.

4. BRIG (BLAST Ring Image Generator) for multiple genome comparison

From Nabil-Fareed Alikhan at the University of Queensland, also as part of a graduate project, which I believe is still in progress…

[ Download | Download BLAST | Paper | Tutorial ]

Fig4_BRIG

BRIG is a free tool that requires a local installation of BLAST (we used BRIG 0.95 on Mac OS X). The output is a static image.

• Launch BRIG and set the reference sequence (EHEC EDL933 chromosome) and the location of other E. coli sequences for comparison. If you include reference sequences for the Stx2 phage and LEE pathogenicity island, it will be easy to see where these sequences are located.

• Click ‘Next’ and specify the sequence data and colour for each ring to be displayed in comparison to the reference.

• Click ‘Next’ and specify a title for the centre of the image and an output file, then click ‘Submit’ to run BRIG.

• BRIG will create an output file containing a circular image like the one shown here. It is easy to see that the Stx2 phage is present in the EHEC chromosomes (purple) and the outbreak genome (black), but not the EAEC or EPEC chromosomes.

Tutorial

The tutorial accompanying the article is available here. To give you an idea of what’s covered, here is the table of contents:

1. Genome assembly and annotation…………………………………………………………… 2

1.1 Downloading E. coli sequences for assembly…………………………………………….. 2

1.2 Examining quality of reads (FastQC)………………………………………………………… 2

1.3 Velvet – assembling reads into contigs………………………………………………………. 4

1.3.1 Using VelvetOptimiser to optimise de novo assembly with Velvet………….. 6

1.4 Ordering contigs against a reference using Mauve………………………………………. 7

1.4.1 Viewing the ordered contigs (Mauve)………………………………………………… 10

1.4.2 Viewing the ordered contigs (ACT)……………………………………………………. 13

1.5 Mauve Assembly Metrics – Statistical View of the Contigs………………………… 15

1.6 Annotation with RAST……………………………………………………………………………. 15

1.6.1 Alternatives to RAST………………………………………………………………………. 19

2. Comparative genome analysis……………………………………………………………….. 20

2.1 Downloading E. coli genome sequences for comparative analysis………………. 20

2.2 Mauve – for multiple genome alignment……………………………………………………. 21

2.3 ACT – for detailed pairwise genome comparisons……………………………………… 24

2.3.1 Generating comparison files for ACT…………………………………………………. 24

2.3.2 Viewing genome comparisons in ACT……………………………………………….. 27

2.4 BRIG – Visualizing reference-based comparisons of multiple sequences……… 29

3. Typing and specialist tools……………………………………………………………………. 34

3.1 PHAST – for identification of phage sequences…………………………………………. 34

3.2 ResFinder – for identification of resistance gene sequences………………………… 34

3.3 Multilocus sequence typing…………………………………………………………………….. 34

3.4 PATRIC – online genome comparison tool………………………………………………… 34