Molecular genetics in China is providing answers in the frantic effort to solve the urgent food safety crisis in EU.
Rapid work in China has applied third generation DNA decoding technologies to decode the German outbreak disease bacterium genome. It has revealed the germ to be a hybrid (which can be described alternatively as a chimera, a true natural GMO). But before readers get excited about what this implies, they need to consider that all E. coli strains are chimeras.
The novel germ has some virulence abilities of a class of pathogenic E. coli bacteria called entero-aggregative E. coli (#EAEC). It has similarities to a bacterial strain called EAEC 55989 , which was isolated in the Central African Republic and is known to cause serious diarrhea. EAEC typically carry extra mini-chromosomes called plasmids. The German outbreak strain has the typical plasmid genes of EAEC bacteria as well as shigatoxin genes seen in EHEC (sometimes called STEC, or VTEC) germs.
The work decoding the genome done in Shenzhen, China, is a triumph of rapid genetic investigation using high-technology methods. The German outbreak strain is a new strain which has acquired specific gene sequences that have a role in pathogenicity, causing hemorrhagic colitis and hemolytic-uremic syndrome (HUS). (See earlier post for some of the ways genes get into E. coli and out again to other bacteria.) Medical articles about EAEC are summarised at the end of this post.
- UPDATE Sep 2011 Technical European Food Safety Agency report that traces the outbreak to E. coli contamination of organic fenugreek seeds grown in Egypt and exported to Europe. Another outbreak with the same germ occurred a few weeks later in France. Detective work on these two (i.e. German and French) common source outbreaks enabled fenugreek seeds to be flagged as the root cause of the infections in both outbreaks. These seeds were imported from an organic farm in Egypt as stated in this report.
- Citation: European Food Safety Authority 5 July 2011. TECHNICAL REPORT OF EFSA
- Tracing seeds, in particular fenugreek (Trigonella foenum-graecum) seeds, in relation to the Shiga toxin-producing E. coli (STEC) O104:H4 2011 Outbreaks in Germany and France
- Worried about deliberate introduction of the German germ? Read this. **(Update on where the strain came from 19-06-2011)**
- Sprouts farm is the suspected culprit
- Out of Germany, not Out of Africa: E. coli strain involved has 10 year history of presence in Germany
- Investigation of the parents to outbreak strain– main points at Biofortified . A re-posting of STEC/EHEC outbreak – horizontally transferred genes
- EHEC: Scientists at University of Münster identify genome sequence of a comparable strain of the year 2001 06.06.2011 Investigations will deliver further indications as to the behaviour of the current pathogen HUSEC041 (O104:H4)
- Real progress on EU E. coli outbreak: An Egyptian fenugreek seed source to European food disaster?
- What Europeans should have read about pathogenic E. coli in 2007 to have helped avoid the current food safety crisis
- Natural GMOs Part 83: Natural biology comes with no guarantee of safety
- The lethal elephant in the room: Real risks in our food
Also read a seriously good and readable update at Nature magazine:
- News: German E. coli outbreak caused by previously unknown strain Genome sequence gives clues to microbe’s sticking power.
European Food Safety Authority:
- Urgent advice on the public health risk of Shiga-toxin producing Escherichia coli in fresh vegetables Published: 09 June 2011. Authoritive, comprehensive timely. EFSA Journal 2011;9(4):2133 [50 pp.]. doi:10.2903/j.efsa.2011.2274
There is a functional annotation of the EHEC coli BGI genome made with our BG7 bacterial genome annotation pipeline running in AWS EC2.
You can get the files HERE
Questions for America at Grist:
Updated discussion about where the chimeric E. coli come from:
A question being raised in many forums is: Do GM foods have a role in the creation of these novel chimeric EAEC E. coli? This is an interesting question, but the answer is almost certainly not.
Consider what David Tribe wrote at Citizendium encylopedia, quoted below.
The bottom line of it is, long before GM crops existed, we knew that bacteria swap genes like crazy, and rates of gene moment were the, and still are, easy to detect with the right methods.
Bacteria swap genes in everybody’s gut whether there is GM food there or not. The GM food issue is irrelevant to this rapid bacterial evolution. All E. coli are chimeras. Besides that, the incriminated seeds came an organic farm in Egypt, and were processed in an organic sprouting facility, so introduction of GM seeds is highly improbable.
Horizontal movement is what bacterial genes do naturally.
The mechanism by which the gene swapping occurred to generate the new German outbreak strain is almost obvious from current knowledge of the biology of gene movement in E. coli. A brief history of this science is explained below, and the role of plasmids (mini-chromosomes) and bacterial viruses mentioned below provide the evidence needed to understand this gene-swapping ability. The following history indicates how this explains the emergence of the German strain. More detail on this science can be found via the link below to Citizendium.
History time line for major discoveries on horizontal gene transfer in bacteria prior to development of GM crops (adapted from D. Tribe at Citizendium; click link)
1928. Discovery of gene transfer in bacteria started in 1928 when Frederick Griffith found Diplococcus pneumoniae bacteria (now usually called Streptococcus pneumoniae) could inherit characteristics (namely the ‘Smooth’ phenotype affecting the bacterial cell coat (capsule)) from non-living extracts of other bacteria. This change was termed transformation. Oswald T. Avery at the Rockefeller Institute in 1944 showed that this transforming material of Diplococcus was DNA.
1946. Joshua Lederberg and Edward Tatum in a series of very carefully designed experiments managed very low-frequency of genetic transfer between Escherichia coli K-12 bacteria when they were in contact with one-another , and this process was later recognized to be caused by the plasmid, fertility factor F.
1951 Joshua and Ester Lederberg , together with Zinder and Lively reported another case of gene transfer that did not need cell-to-cell contact. In this case the bacteria were Salmonella and it was later demonstrated that a bacterial virus (bacteriophage) was carrying the genes between cells, and this is now called transduction.
[Note: Transduction is the likely mechanism for creation of the new German strain from parental EAEC and EHEC parental strains. The shigatoxin gene known to be present in the German outbreak strain , called stx2, is usually carried in a bacterial virus (=bactereriophage or phage in technical jargon) as a set of extra genes inserted in the EHEC genomes. [A later post provides evidence that this is the case in the outbreak strain]. Such inserted virus DNA packages, called prophages, are known to be genetically mobile, and the German outbreak strain most likely has gained a stx2 prophage by infection with a stx2 virus released from a EHEC bacterium. This mechanism of creation is quite predictable from standard knowledge of bacterial genetics]
1960s. DNA transformation was demonstrated in a wide variety of bacterial species including species of Streptococcus, Haemophilus, Neisseria, Bacillus, Synechococcus cyanobacteria, and Rhizobium. Later studies showed that is usually a highly evolved complex biological adaptation for genetic exchange, and, for instance, in Bacillus depends on inter-cellular signaling and Quorum sensing processes and is triggered by stress.
Transmissible plasmids in microorganisms
1950-1958. Conjugation in E. coli is shown to be a one way (F+ donor to F- recipient), process, and not two-way cell fusion. The ability to do this is encoded by a DNA containing factor F 250,000 base pairs in size, whose transfer converts recipients to capability of doing further DNA donation, and which can move infectiously through a recipient bacterial population. Simply, put, sex is infectious in bacteria. F factor was found to be able to move by conjugation to different species such as Salmonella.
1958. The existence of several genetic structures that can insert within bacterial chomosomes, based on observation of the bacteriophage lambda and fertility factor in 1958 F, lead Francois Jacob and Elie L. Wollman  to coin the term episome for DNA elements that have alternate modes of existence within the cell, either in the chromosome, or as autonomously replicating structures. Subsequent study of these phenomenon revealed numerous occurrences of mobile DNA in a wide range of organisms (such as the presence of insertion sequence (IS) ‘jumping genes’ (discussed further below) that allow F plasmid insertion in the chromosome) and widespread horizontal gene transfer involving by bacteriophage, plasmids and mobile DNA in general.
1959. Tomoichiro Akiba and Kunitaro Ochia discover mobile antibiotic resistance genes in bacteria , and the horizontal transfer of antibiotic resistance traits is later shown to mediated by plasmids that inject DNA promiscuously into other cells .
[Note: Plasmids are know to carry some of the disease-related genes in EAEC. The name of the plasmid in EAEC strains of E. coli is pAA, and this is mentioned in the medical science papers at the end of the main post. Some plasmids can move between cells of different species at a relatively high frequency. Genome DNA evidence shows similar plasmids are present in the German outbreak strains.]
1964. Brinton establishes that F factor containing bacteria have pili (fimbriae) fiber-like appendages that are involved making contact with recipients to they can transfer DNA. Similar pili are specified by some R-plasmids (see Pilus).
1960s. R-plasmids are established to be promiscuously transferred among different enteric bacteria. Typically plasmids can be transferred easily among the different species of common gut Gram negative bacteria. Transmissible plasmids are common in stool bacteria, and typically 10-20% of gut E. coli of healthy people (1960-1970) have R-plasmids and are antibiotic resistant. Bacteria are discovered to produce colicins, lethal compounds that kill other bacteria which are specified by transmissible plasmids similar to F plasmid. Examples are ColE, ColV plasmids.
It is also fully realized that F-factor is not unique, but a special case of a very general phenomenon in bacteria of transmissible and mobile DNA, including R-plasmids, colicin determining (Col) plasmids, and prophages. Interactions between them were discovered, for instance R-plasmids were found which interfered with F-fertility. Plasmids were also discovered that carried bacterial virulence genes, Hemolysin (Hly), and surface antigens (K88). Plasmids are shown to be closed circular DNA molecules which are much smaller than the generally circular bacterial genome.
Insertion sequences (IS), transposons (Tn) and Mu
1968. James Shapiro discovers that spontaneously occurring insertions of large inserts of extra DNA can causes mutations in the galactose genes of the bacterium Escherichia coli . This discovery ultimately led to the discovery of mobile insertion sequences (IS). The process of movement of DNA into an new location on a chromosome from an old location is referred to by geneticists as transposition. Generally mobile DNA is often called a transposon and IS are the simplest example. The similarity of IS transposition to mobile (transposon) genes studied much earlier by Barbara McClintock in maize was immediately realized. Transposable mobile DNA is frequently a component of natural plasmids . F-factor carries more than one IS element, and they (as Tn1000) are implicated in enabling bacterial chromosomes to take part in F-mediated conjugation, or mating between different bacterial strains and species. Plasmids and transposons provide numerous mechanisms that allow horizontal gene movement between bacterial species, and facilitate diverse chromosome rearrangement that are thoroughly studied by many microbiologists. Their role in natural gene movement between various human and animal gut bacteria in the guts of living animals is thoroughly demonstrated.
1963-1995. Mutator phage Mu of E. coli which inserts at numerous different sites in the E. coli chromosomes as a prophage, and replicates vegetatively by repeated transposition, provides a convenient tool for elucidation of the mechanisms of transposition in great detail  . Transposition is usually a rare event, and is difficult to analyze biochemically with most transposons and insertion sequences. The more frequent “jumping gene” activity of Mu phage facilitated its elucidation as an understandable chemical reaction of DNA.
1974. The first transposition of a gene for ampicillin resistance from one R-plasmid to another plasmid is fully characterized . The ampicillin resistance transposon is named Tn3. Other transposons are named Tn5 (kanamycin resistance with IS50 at each termini) , and Tn10 ( tetracycline resistance). R-plasmids are currently thought of as modular replicons put together with Tns and replication origins (ori sites ) providing plasmid-specific sites for initiation of DNA replication, and transposition of Tns is seen to be part of plasmid natural evolution.
Details of the Chinese work quoted above:
- REPOSTING OF BGI ANNOUNCEMENT BGI Sequences Genome of the Deadly E. coli in Germany and Reveals New Super-Toxic Strain: Genome sequence identifies super-toxic bacteria as cause of the current European epidemic. June 2nd, Shenzhen, China
The recent outbreak of an E. coli infection in Germany has resulted in serious concerns about the potential appearance of a new deadly strain of bacteria. In response to this situation, and immediately after the reports of deaths, the University Medical Centre Hamburg-Eppendorf and BGI-Shenzhen began working together to sequence the bacterium and assess its human health risk. BGI-Shenzhen has just completed the sequence and carried out a preliminary analysis that shows the current infection is caused by an entirely new super-toxic E. coli strain.
According to the latest announcement from German health officials, the death toll in Europe from the epidemic has risen to at least 17. Over 1,000 new cases of infection have also been reported in other parts of Europe, including Sweden, Denmark, the Netherlands, the UK, and others. The University Medical Center Hamburg-Eppendorf received the majority of the infected patients from northern Germany and found that antibiotic treatment was ineffective.
BGI was informed of the dangerous situation and, in collaboration with the University Medical Center Hamburg-Eppendorf researchers, used their genomic technology to determine the infectious strain, reveal the mechanisms of infection, and facilitate the development of measures to control the spread of this epidemic.
Upon receiving the bacterial DNA samples, BGI finished sequencing the genome of the bacterium within three days using their third-generation sequencing platform — Ion Torrent by Life Technologies. Bioinformatics analysis revealed that this E. coli is a new strain of bacteria that is highly infectious and toxic.
According to the results of the current draft assembly (available for download at ftp://ftp.genomics.org.cn/pub/Ecoli_TY-2482), the estimated genome size of this new E. coli strain is about 5.2 Mb. Sequence analysis indicated this bacterium is an EHEC serotype O104 E. coli strain; however, this is a new serotype — not previously involved in any E. coli outbreaks. Comparative analysis showed that this bacterium has 93% sequence similarity with the EAEC 55989 E. coli strain, which was isolated in the Central African Republic and known to cause serious diarrhea. This new strain of E. coli, however, has also acquired specific sequences that appear to be similar to those involved in the pathogenicity of hemorrhagic colitis and hemolytic-uremic syndrome. The acquisition of these genes may have occurred through horizontal gene transfer. The analysis further showed that this deadly bacterium carries several antibiotic resistance genes, including resistance to aminoglycoside, macrolides and Beta-lactam antibiotics: all of which makes antibiotic treatment extremely difficult.
The research team will further analyze the integrity of the virulence genes, their expression profiles, drug resistance, and gene transfer mechanisms followed by validation of these genes in other strains. In addition BGI and collaborators are developing diagnostic kits to aid in curtailing this epidemic. New results will be continuously updated. (To obtain immediate updates see Twitter: @BGI_Events.)
The sequences of this new E. coli strain have been uploaded to NCBI (SRA No: SRA037315.1) and are also available for immediate download at ftp://ftp.genomics.org.cn/pub/Ecoli_TY-2482.
About BGI: BGI (formerly known as Beijing Genomics Institute) was founded in 1999 and has since become the largest genomic organization in the world. With a focus on research and applications in the healthcare, agriculture, conservation and bio-energy fields, BGI has a proven track record of innovative, high-profile research which has generated over 178 publications in top-tier journals such as Nature and Science. BGI’s distinguished achievements have made a great contribution to the development of genomics in both China and the world. Our goal is to make leading-edge genomics highly accessible to the global research community by leveraging industry’s best technology, economies of scale and expert bioinformatics resources. BGI and its affiliates, BGI Americas and BGI Europe, have established partnerships and collaborations with leading academic and government research institutions, as well as global biotechnology and pharmaceutical companies. At BGI, we have built the infrastructure and scientific expertise to enable our customers and collaborators to quickly migrate from samples to discovery. For more information, visit http://www.bgisequence.com
FURTHER TECHNICAL DETAILS RELATING TO THE NEW GERMAN E. coli DISEASE OUTBREAK STRAIN
More about EAEC: A Review
- Enteroaggregative Escherichia coli: An Emerging Enteric Food Borne Pathogen by P. Kaur, A. Chakraborti, and A. Asea in Interdiscip Perspect Infect Dis. 2010; 2010: 254159. Published online 2010 March 11. doi: 10.1155/2010/254159. PMCID: PMC2837894
Abstract: Enteroaggregative Escherichia coli (EAEC) are quite heterogeneous category of an emerging enteric pathogen associated with cases of acute or persistent diarrhea worldwide in children and adults, and over the past decade has received increasing attention as a cause of watery diarrhea, which is often persistent. EAEC infection is an important cause of diarrhea in outbreak and non-outbreak settings in developing and developed countries. Recently, EAEC has been implicated in the development of irritable bowel syndrome, but this remains to be confirmed. EAEC is defined as a diarrheal pathogen based on its characteristic aggregative adherence (AA) to HEp-2 cells in culture and its biofilm formation on the intestinal mucosa with a “stacked-brick” adherence phenotype, which is related to the presence of a 60 MDa plasmid (pAA). At the molecular level, strains demonstrating the aggregative phenotype are quite heterogeneous; several virulence factors are detected by polymerase chain reaction; however, none exhibited 100% specificity. Although several studies have identified specific virulence factor(s) unique to EAEC, the mechanism by which EAEC exerts its pathogenesis is, thus, far unknown. The present review updates the current knowledge on the epidemiology, chronic complications, detection, virulence factors, and treatment of EAEC, an emerging enteric food borne pathogen.
- Enteroaggregative Escherichia coli by James P. Nataro, Theodore Steiner, and Richard L. Guerrant (1998) Emerging Infectious Diseases Vol 4 April-June 1998 (Early review but free access with images)
- Typical Enteroaggregative and Atypical Enteropathogenic Types of Escherichia coli Are the Most Prevalent Diarrhea-Associated Pathotypes among Brazilian Children by Joel M. Araujo, Graciela F. Tabarelli, Katia R. S. Aranda, Sandra H. Fabbricotti, Ulysses Fagundes-Neto, Caio M. F. Mendes, and Isabel C. A. Scaletsky in J Clin Microbiol. 2007 October; 45(10): 3396–3399. Published online 2007 August 1. doi: 10.1128/JCM.00084-07. PMCID: PMC2045331
Abstract: A 1-year prospective study was carried out in two large urban centers of São Paulo State, Brazil, to determine the prevalences and roles of the different Escherichia coli pathotypes in children less than 5 years of age with diarrhea presenting to the emergency rooms of public hospitals or visiting private pediatricians’ offices. Of the pathotypes sought, typical enteroaggregative and atypical enteropathogenic types of E. coli were isolated for 8.9% and 5.4% of 774 diarrhea cases, respectively, and were found to be dominant and significantly associated with diarrhea.
- High prevalence of aggregative adherent Escherichia coli strains in the mucosa-associated microbiota of patients with inflammatory bowel diseases. by Thomazini CM, Samegima DA, Rodrigues MA, Victoria CR, Rodrigues J. from Institute of Biosciences, Department of Microbiology and Immunology and Faculty of Medicine, Department of Pathology, Botucatu, SP, Brazil. in Int J Med Microbiol. 2011 May 25. [Epub ahead of print]
Abstract: The intestinal population of Escherichia coli is increased in patients with inflammatory bowel disease (IBD), but the reason for this elevation, the particular features of these bacteria and their potential role in the pathogenesis of the disease are not known. The present study was undertaken to investigate the adherence abilities and some virulence properties of a collection of 131 E. coli isolates cultured from rectal biopsies of 23 subjects diagnosed with ulcerative colitis (UC), 8 with Crohn’s disease (CD) and 23 control patients from southern Brazil. The adherence abilities of the bacteria were investigated in vitro, using HEp-2 epithelial cells in assays of 3 and 6h of bacteria-cell contact. The isolates were screened by PCR with primers for the following virulence genetic markers: plasmid of aggregative adhesion (pAA) and the aggregative adherence fimbriae R (aggR), E. coli attaching and effacing (eae), invasion-associated locus (ial), invasion plasmid antigen H (ipaH) and Shiga citotoxin-encoding (stx) genes. HEp-2 cells aggregative adherent E. coli strains, as detected in the 3h adherence assay, were found in 14/23 (60.9%) patients with UC, 7/8 (87.5%) with CD and in 7/23 (30.4%) controls (p=0.011). Virulence genetic markers were detected in strains of 9 patients with UC (39.1%), but in none of CD or control group. Two of these UC patients had strains harboring both pAA and aggR, one had strains positive for aggR, four had strains positive for eae and two had strains positive for stx. These results suggest that the augmented population of E. coli on the rectal mucosa of IBD patients, particularly of those diagnosed with UC, is mostly comprised of aggregative adherent strains, some of which possessing classical virulence markers of E. coli.
- Single multiplex assay to identify simultaneously enteropathogenic, enteroaggregative, enterotoxigenic, enteroinvasive and Shiga toxin-producing Escherichia coli strains in Brazilian children. by Aranda KR, Fabbricotti SH, Fagundes-Neto U, Scaletsky IC from Departamento de Microbiologia, Imunologia e Parasitologia, São Paulo, SP, Brazil. In FEMS Microbiol Lett. 2007 Feb;267(2):145-50. PMID: 17328113
Abstract: A multiplex PCR to differentiate typical and atypical enteropathogenic Escherichia coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic (ETEC), enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC) strains was developed and evaluated. The targets selected for each group were eae and bfpA for EPEC, aggR for EAEC, elt and est for ETEC, ipaH for EIEC and stx for STEC isolates. This PCR was specific and sensitive for rapid detection of target isolates in stools. Among 79 children with acute diarrhea, this technique identified 13 (16.4%) with atypical EPEC, four (5%) with EAEC, three (3.8%) with typical EPEC, one (1.3%) with ETEC and one (1.3%) with EIEC.
- Is this e-coli strain a mutation and how does this affect the current outbreak? 2 June 2011 on risks from Escherichia coli (from ECDC)
The WHO has recently stated that this strain of e-coli “is a unique strain that has never been isolated from patients before” and there may be “various characteristics that make it more virulent and toxin-producing”.
The WHO reference laboratory has been able to confirm that this appears not to be a classical STEC. However, some reports of mutation are incorrect. In simple terms “mutation” refers to a modification of a gene whereas the current issue raised by the WHO is better explained as a matter of acquiring extra genes or natural genetic recombination which is often seen with bacteria.
This modification is slightly more unusual, and thus deserves our attention, as it involves strains of e-coli that are not often associated. However this is an important piece of information and will help all the authorities involved in this outbreak by providing extra information on the outbreak strain characteristics. This is important as it will allow us all to better understand the clinical and epidemiological behaviour of this pathogen compared to disease caused by other STEC strains. It is important to note, however, that this at present does not change the current epidemiological information which suggests the outbreak is still focussed on Northern Germany.
Public health advice on prevention of diarrhoeal illness with special focus on Shiga toxin – producing E. coli (STEC). A joint statement by the European Centre for Disease Prevention and Control (ECDC) and the European Food Safety Authority (EFSA), 1 June 2011.
- Risk assessment on Escherichia coli (STEC) outbreak in Germany 27 May 2011 (PDF)
ECDC has conducted a rapid risk assessment following the unusual increase of Shiga toxin-producing Escherichia coli (STEC) infections in Germany, with patients presenting with haemolytic uremic syndrome (HUS) and bloody diarrhoea.