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--A recent study uses a new approach to investigate the shiga toxin producing bacteria responsible for a serious
disease outbreak in Germany in 2011. The real culprits behind the outbreak are the viruses that carry
the gene for shiga toxin and transfer it to otherwise harmless bacteria --
What’s
harder than finding a needle in a haystack? Finding the bacterial genome you’re
looking for in a diarrhea sample. A recent study
published on April 10, 2013 in the Journal of the American Medical Associaton (JAMA)
made this task seem relatively easy. The bacteria being searched for was a rare shiga toxin producing
bacteria that causes bloody diarrhea and other severe complications in
humans upon infection. This study was done by an international team of
researchers coordinated by Mark
J. Pallen who recently became the head of Warwick Medical School’s new Division
of Microbiology and Infection. The bacterial strain that caused an outbreak in Germany was especially rare making it hard to identify. Because of this, researchers employed a new method to
identify the genome sequence of this highly pathogenic bacteria. Their method of detection was to sequence all the genetic
material present in fecal samples from patients with diarrhea during the outbreak
and sort through this genetic information to find the sequence of the disease causing strain.
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The source of the outbreak in Germany during the summer of 2011 is believed to be from the consumpton of raw
sprouts contaminated with the dangerous bacteria strain (2). This outbreak affected
thousands of people in a wealthy, modern, industrialized society, causing more
than 50 deaths (4). In times like this, quick identification of
the causative pathogen (in this case a shiga toxin producing bacterial strain)
is critical for the management of the outbreak. Traditionally, the standard for identifying
pathogens in clinical samples is to isolate the disease causing bacteria from
other microbiota in the samples and then sequence it once it is in pure
culture. This study's approach is different becuase they directly sequenced the mixed communities of bacteria and anything else present within the feces sample and then analyzed the sequence data to find
the disease causing bacteria. The sequencing of mixed microbial communities is called metagenomics
and allows identification independent of laboratory isolation and culture of the causative
bacteria.
To
extract DNA, 45 fecal samples were taken from patients with diarrhea during the
2011 outbreak. After getting the sequence of the genomes in the fecal samples, bioinformatics
was used in order to sort through and piece together the genome of the pathogenic
strain of bacteria. The goal was to find what strain of bacteria all the infected people had in common. This involved a series of steps starting with screening out
stretches of genomes that are known to be human DNA sequences. Next, the microbial genes were assembled into a collection of “environmental gene
tags” (EGTs). EGT’s are short sequences of DNA that can be used to identify or
characterize the organisms from which they come from. They also compared samples from
healthy individuals to rule out bacteria present in those samples. Once the
EGTs in common with the healthy individuals were discarded, just 450 outbreak-specific
EGT’s were left in common between affected patients. Of these EGT’s, 65% were assigned to the Enterobacteriales, the
order that contains E. coli. These
EGT’s were used to reconstruct genome of the outbreak strain. Not all E. coli are pathogenic so it was
important to identity what accessory genes were present in the E. coli strain that caused it to be
harmful. One such accessory gene that was important in this outbreak is the shiga-toxin gene.
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Figure 1. Bacteriophage infecting their host E. coli
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The
focus here is on the bacteria, but in order to understand the pathogenicity of this
strain I’d like to bring attention back to the viruses that serve as their
accomplices. Many of the E. coli that reside in our gut are not harmful. In
order to become disease causing, the E.
coli must acquire a combination of genetic elements . One of the most
important changes that needs to happen is the ability to produce the shiga toxin.
The gene coding for the shiga toxin does not come from the bacteria itself – it
is a gene encoded by viruses that infect the bacteria (bacteriophages). So in
order for the bacteria to be disease causing it needs to be infected by a bacteriophage
(phage) that carries the shiga toxin gene.
Interestingly,
this study found an unexpected copy number of shiga-toxin genes compared to
other chromosome loci of the shiga-toxigenic Escherichia coli (STEC) outbreak
strain. Overall there more shiga toxin encoding regions in the samples positive for STEC than expected. However this varied from sample to sample
that were positive for the outbreak strain. Some patient samples had more and
some had less of the phage genome relative to bacterial genome pieces. The researchers were not sure why this was
the case. They speculate that this could have to do with detection of phage
particles that are released from the bacteria upon the lytic phase of their life
cycle. This also could be due to multiple phage insertions or duplications
within individual E. coli
genomes. Further studies are
needed in order to clarify what is going on with the transfer of phage genomes
in these bacteria and thier impact on disease. Further studies would be beneficial because it relates to
treatment of this disease. Studies have found that antibiotics may actually be
helping the shiga toxin viral genes to spread. When bacteria are exposed to certain types of antibiotics
they undergo a stress response, which induces phage replication within the
bacteria. Upon active replication, the bacteria cell bursts open and releases
the newly synthesized viral particles. This also releases the toxin, which is
why antibiotics are not used to treat these infections (4).
This
metagenomics approach is an interesting one and in some ways superior to
previous techniques used to identigy pathogens. The general method used for identifiying an
outbreak strain involves isolating the bacteria in pure culture and then
sequencing it. Metagenomics skips the step of in vitro culture by directly sequencing the mixed communities present within the feces sample. Previous in vitro methods of
isolating the strain in pure culture can be slow difficult or even impossible
to indentify certain strains. This
becomes especially difficult when the outbreak strain is very rare like the outbreak stain in Germany in this study (5). This metagenomic
approach is important because there actually is no single in vitro microbioloical test accepted as an official
standard to detect STEC.
However
this metagenomics approach is not bullet proof and continuous research is
needed to improve it. The researchers of this study admit that this approach
does not guarantee to find the single causative pathogen of the disease.
Multiple pathogens were found in some of the samples and it was impossible to
conclude with 100% certainty which of those is the cause of the disease. Another thing to keep in mind is that genome sequence without any information of function or about what species it is coming from is of little use. That is why more in vitro experiments
are still needed so that we can continuously gain more functional information about the genes like those causing E. coli to be pathogenic. This metagenomic approach is also currently expensive and requires
a lot of data anaylsis and processing. However researchers are optimistic because with the improvement of sequencing technology and the increase of annotated genetic information in databases these setbacks can be overcome.
Overall, using metagenomics, the direct sequencing of mixed microbial
communities, is a very promising approach for identifying pathogens. The genomic sequence of the pathogen that
causes an outbreak is very important for managing the outbreak. The fecal samples from patients have a mix of genomic data within
them, human, viral, and bacterial. Sorting though all the mixed genomic data
within a clinical sample is not easy, but this study shows it can be done
successfully to isolate sequence of even a rare pathogen. What do you get when you mix metagenomics and poop samples?
Lots of information! So we should all start ‘giving a poo’, for the sake of
science.
After thought: Other
applications of Metagenomics
The
applications of metagenomics do not stop here at identifying outbreak strains
of bacteria. While much clinical emphasis has been put on the microbes (bacteria, viruses, fungi, and protozoa) that cause disease, there
are many microbes that contribute to our health like the bacteria in our gut
that help us with digestion. Metagenomics is a powerful method to assess the
microbiota which we are finding to be more and more important for our health.
We already know that our microbial flora (those that call our body their home)
out number our own cells 10-fold with a total gene content of 100-fold greater
than our own genome. To find out
more about why it has become fashionable to sequence poop, read the
paper written by Harvard Professor Christopher Marx about the application
of metagenomics for evolutionary studies (6).
Primary Article
1. Loman, N.,
Constantinidou, C., Christner, M., Rohde H., Chan, J., Quick, J., Weir, J.,
Qince, C., Smith, G., Betley, J., Aepfelbacher, M., Pallen, M. (2013) A Culture-Independent
Sequence-Based Metagenomics Approach to the Investigation of an Outbreak of
Shiga-Toxigenic Escherichia coli
O104:H4. JAMA , 309(14): 1502-1510.
Supplementary sources
3.Viruses can turn harmless E. coli
dangerous (2009) Science Daily: http://www.sciencedaily.com/releases/2009/04/090417195827.htm
4. Turner, M (2011) Antibiotic use may have driven the development of Europe's deadly E. coli. :
5. Bloch, S., Felczykowska, A.,
Nejman-Falenczyk, B. (2012) Escherichia coli O104:H4 outbreak – have we learnt
a lesson from it?
6. Marx, Christopher (2013) “Can You
Sequence Ecology? Metagenomics of Adaptive Diversification” http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001487
Really enjoyed the discussion on metagenetics. I think that it is a really interesting research technique and I agree with the applications you mentioned. I didnt know much about it and this article really helped me out
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