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June proved to be a busy month con-cerning epidemics in Europe. Before May came to a close, nine countries, led by Germany, reported about a thousand cases of enterohaemorrhagic Escherichia coli (EHEC) infections and increasing numbers of haemolytic uraemic syndrome (HUS). By the end of July, the EHEC strain 0104:H yielded more than 3, 800 confirmed infections and 823 cases of HUS, with 44 deaths. HUS proved to be lethal again causing acute renal failure and a low platelet count predominantly affecting children. Thus, EHEC is a deadly version of the E. coli bac-terium usually found in the gut of humans and animals. For a continent known for careful handling of food, this came as a shock. But it was not only startling news in terms of food contamination. How the outbreak was handled was appalling. Vari-ous vegetables were blamed for the disease. The manner of outbreak communication and handling also received critical response. This is FEMS Focus’ take on communicating the EHEC crisis.

Tone Tønjum & Chared Verschuur-Ballo,

Editors

What is your role in the E. coli research?

Dr Helge Karch (HK): We focus on en-terohemorrhagic E. coli (EHEC) which can cause, in addition to diarrhoea, sys-temic complications, most notably the haemolytic uraemic syndrome (HUS). Our work addresses several broad, but interacting aspects of human infec-tions: where do EHEC reside when they are not infecting humans? how do they cause human disease? how do they evolve and adapt to human and non-human hosts and to environment milieus? which virulence traits are in-volved in transmission, adaptation and pathogenesis, and what is the basis for each of these mechanisms?To answer these questions, we analyze the epidemiology, diversity, phylogeny, and function of virulence factors from EHEC. Currently, we are most actively studying various alleles of Shiga toxins (Stxs), the EHEC-hemolysin, coloniza-tion factors including the Sfp fimbrial adhesin, and a family of immunoglobu-lin-binding proteins.Current understanding strongly sug-gests that Stxs are the predominant viru-lence factors in injury of microvascular endothelial cells in the kidneys and the brain, which cause the clinical picture of HUS. Therefore, we are characterizing the molecular assembly of the Stx-gly-cosphingolipid receptors in the plasma membrane microdomains (lipid rafts) of these target cells. Such studies will provide data on the initial molecular interaction of Stxs with the human en-

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To communicate the EHEC crisis effectively, FEMS Focus interviewed two experts on the bacterium. Dr Helge Karch is a leading German E. coli researcher who discovered what features made the bacteria responsible for the outbreak particularly pathogenic. He has been working with diarrheagenic E. coli since 1983 as part of his postdoctoral training. He is currently the Director of the Institute of Hygiene at the University Hospital Münster.Dr Miguel Vicente is a molecular biologist by training. Over the years, he developed an interest in pathogens. Currently based at Centro de Investiga-ciones Biológicas (CIB) at CSIC in Madrid, his first contact with E. coli research was in 1973 during a long-term fellowship.

EUROPE’S E. COLI CRISIS

Microbes turn wildFrom the Editorial Team

dothelium, the prerequisite for binding and internalization of Stxs. This would be an ideal opportunity for therapeutic intervention in infected hosts. Another important part of our research activi-ties focuses on mechanisms involved in evolution of these strains in the en-vironment and during infection. These analyses include functional analysis of pathogen-host interactions as well as systematic and thorough population- and geographic interrogation of micro-bial molecular epidemiological markers.

Dr Miguel Vicente (MV): I am fascinated by how bacteria, apparently very mod-

est organisms, manage to regulate so precisely their growth and their proliferation. How they coordinate, both in time and space, the divi-sion process with their growth rate involves extremely accurate mechanisms that after all these years, and thanks to the efforts of many groups working in this topic, we are just on the verge of understanding. Knowledge on the bac-terial cell division cycle has been largely derived

Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded or-ganisms. Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans. One of these strains is the O104:H4 that caused the 2011 out-break. Source: Wikimedia Commons

from the study of K12, a harmless laboratory strain of E. coli, and its many derivatives. At present they include mutants in most of the genes whose products are essential for the processes of chromosome replication, nucle-oid segregation and septation. My group has contributed to describe the role of some of these products in the assembly and operation of the septation machinery. We have also in-vestigated how the genes that encode a large number of them are grouped in a chromo-somal region. This region is conserved in rod-shaped bacteria and the expression of its genes is controlled through complex signals that en-sure the production, at any growth rate, of one septum per cell. Recently we are attempting to reconstruct parts of this machinery in the test tube hoping that we will be able to reproduce their function outside the bacterial cell.

What can you say about the current EHEC outbreak? What are the characteristics of the current EHEC outbreak?

HK: Such a virulent EHEC strain as the O104:H4 outbreak strain of the HUSEC041 clone complex has never before challenged the human population and medical science. With hundreds HUS cases and at least four dozen deaths it is the most terrible outbreak of HUS in history. To analyze this tragedy, we rapidly mobilized field epidemiology, molec-ular typing, and high-throughput genomics to an unprecedented extent. This outbreak is most unusual in its case attack pattern: most patients are adults, mainly younger women, which is in contrast to outbreaks caused by the prototypic EHEC O157:H7 affecting mainly children and the elderly persons (> 65 years). There appears to be a disproportion-ate frequency of severe neurological compli-cations, such as encephalopathy and epileptic seizures. The reasons for the atypical age dis-tribution and the special clinical features as well as the origin of the outbreak strain are largely unknown, though it does suggest a vehicle other than ones that more commonly transmit EHEC O157:H7.

How virulent is the HUSEC041 clone that is causing the current EHEC epidemic as compared to other EHEC strains and to the normal E. coli?

HK: The outbreak strain seems to be extraor-dinarily virulent as indicated by the unpre-cedented number of patients that developed HUS and then died. The reasons for the apparent-ly increased viru-lence of the out-break strain is yet unknown and is the subject of ongoing research in my labora-tory. As we have demonstrated in our recent publications one reason can be that the strain efficiently combines major virulence charac-teristics of EHEC and enteroaggregative E. coli (EAEC), i.e. production of Stx and abil-ity to intensively adhere to intestinal epithe-lial cells in an aggregative manner typical for

EAEC. We hypothesize that the strong adher-ence phenotype enhances systemic absorption of Stx produced by the colonizing bacteria; this, indeed, could increase the rate and se-verity of systemic complications (HUS), be-cause more toxin can (theoretically) bind the glomerular microvasculature, the major target affected by Stx during HUS. This pathogen is much more adherent than EHEC O157:H7.

MV: In contrast to the E. coli K12 laboratory strains, and to the commensal strains of the gut, the pathogenic E. coli clones contain genes that encode nasty proteins, those that cause disease in humans and animals. The EHEC strains cause a severe bloody diar-rhoea. In plain words, they damage the cells that line our intestine, sitting on their top, modifying their membranes and injecting proteins into their interior that subvert and

deteriorate their physiology. The O104:H4 strain, isolated from the patients of this outbreak, pos-sesses other un-

desirable traits. One is a gene that codes for a toxin, called Shiga, that ends up in the blood-stream, acting mostly within the capillaries of smallest diameter. The Shiga toxin even alters some components of the immune system and it has a final deleterious effect on the mal-functioning of the filtering devices present in the kidneys. This is the reason why, when the

E. coli, EAEC in brief. This came as a surprise because, after sequencing its genome in full, it seemed to lack the gene for intimin, one of the proteins that contributes to the efficient attachment of other EAEC strains.

How resistant are the current E. coli outbreak strains to antibiotics?

HK: The outbreak strain has an extended spectrum beta-lactamase (ESBL) phenotype, i.e. it is resistant to all penicillins and cepha-losporins and susceptible to carbapenems (ertapenem, imipenem, meropenem). Fur-thermore, all outbreak isolates analysed in our laboratory are resistant to trimethoprim/sulfamethoxazole and susceptible to fluoro-quinolones (ciprofloxacin) and aminoglyco-sides (gentamicin, tobramycin).

MV: E. coli is a Gram-negative bacteria, mean-ing that it has an envelope formed by a rigid layer of peptidoglycan sandwiched in be-tween two membranes. This structure makes

all E. coli strains more resistant to beta-lactam antibiotics, like penicillin, that interfere with the integrity of the peptidoglycan. However, the use of beta-lactams to treat EHEC infec-tions would provide a mixed blessing, as they cause bacterial lysis and consequently the re-lease of free toxin into the patient organism. Other antibiotics, as quinolones, that inter-fere with DNA replication, may also have the undesired effect of increasing the amount of toxin produced by STEC strains due to the increased expression, or even the increased number, of the toxin genes in their presence. These are among the reasons why antibiotics are a rather weak choice to fight diarrhoea caused by these E. coli strains. The O104:H4 outbreak strain additionally contains a battery of resistances against several antibiotics which are not uncommon nowadays among the clinical isolates of E coli. It also contains re-sistance against heavy metals. On the positive side, it appeared to be sensitive to carbapen-ems, a class of beta-lactams used in hospitals, less prone to be destroyed by the betalacta-mase activity present in many E. coli strains.

Should we be afraid that this can be used for bio-warfare?

HK: Like any highly virulent pathogen, the EHEC O104:H4 outbreak strain could be used as a potential bio-weapon.

MV: Contamination by E. coli most frequent-ly occurs through the food, drinks or water supply that we consume. The regulations that apply to food safety and water treatment are usually effective in avoiding infection. It is only in cases in which accidentally some of these control measures fail that infectious outbreaks occur. These regulations should be equally efficient to avoid the spread of pathogens independently of their origin, what makes the use of this strain for crimi-nal purposes unlikely. In addition, cooking

Colonies of Escherichia coli bacteria grown on a Hektoen enteric (HE) agar plate mediumSource: CDC

Helge Karch determined the type HUSEC041 of the EHEC-intestinal bacterium this year (2011). Currently the Director of the Institute of Hygiene, University Hospital Münster, his team was the first who identified the recent outbreak strain on May 25, 2011 and developed a test for its specific identification five days later. This information was delivered to the public on May 30, 2011 via www.ehec.org. They were the first who made the complete sequence of an outbreak strain available on June 3rd and published (Lancet Infectious Diseases, June 22nd 2011, Epub ahead of print) a complete microbiological analysis of the HUSEC041 complex including eighty 2011 outbreak isolates and a historical O104:H4 isolate which they isolated from a HUS patient in Cologne in Sept. 2001.

Source: Helge Karch

Miguel Vicente is Professor of Research at “Centro Nacional de Biotecnología (CNB-CSIC)” in Madrid, where he leads a group working on the proliferation of E. coli and other bacteria. His group focuses on bacterial cell division, to describe the process of assembly of the division proteins, the stabil-ity of the protein ring and the interaction between the different components (www.cnb.uam.es/~mvicente/index.html). Trained in Molecular Microbiology, he presently coordinates an EC FP7 project, DIVINOC-ELL, gathering groups from eleven European and South American institutions aiming at discovering new drugs to combat infections caused by Gramnegative bacteria.

Source: Inés poveda

procedures involving heating and boiling, or peeling and disinfecting, in the case of food to be eaten raw, destroy the viability of E. coli.

Has an antidote / antibody to the toxin been developed?

HK: Antibodies to the Shiga toxins were devel-oped just after the characterization of the tox-ins in the late 1980’s. Several antibodies have been tested in animal models, but only two in humans. Actually there is only one left in ac-tive clinical development, Shigamabs™, chi-meric antibodies to Stx1 and Stx2. This pro-gram is in phase II testing in South America.

MV: The severity of this outbreak brought to light the existence of an experimental drug, an antibody produced under the name ecu-lizumab that was being developed to treat paroxysmal nocturnal haemoglobinuria, a seemingly unrelated disease. It seems that the antibody blocks some components of the immune system, the activation of the C5 protein of complement. This activation, leading to cell damage and tissue destruction, also occurs in the uremic syndrome caused by the STEC strains. Eculizumab had already been used successfully to treat some cases of uremic syndrome infections. By blocking C5 activation, the destruction of capillary tissue is alleviated, bringing relief to some patients.

What can you say about the way this outbreak has been handled and communicated?

MV: Driving to my lab this morning, I was listening to my favourite radio station while a discussion took place on how the issues related to food safety are communicated to the Spanish public. The speakers, including one journalist, one doctor and one consumer advocate agreed that in this case, named the “cucumber crisis” in Spain, the information to the public had been correct. Having been busy with the media for almost one week,

This map depicts the 2011 E. coli O104:H4 out-break. Source: Wikimedia Commons

organs that could otherwise remove the toxin stop working, some patients develop HUS, and their infection may become fatal. The strains that produce this kind of toxin are clas-sified as STEC, meaning Shiga toxin E. coli.This strain has also proven to be very effec-tive in attaching to the surface of the intesti-nal cells, making it more dangerous because the infection stays longer. Consequently, it has also been classified as enteroaggregative

“By the end of July 2011, the enterohaemorrhagic Escherichia coli EHEC strain 0104:H yielded more than

3, 800 confirmed infections and 823 cases of haemolytic uraemic syndrome (HUS), with 44 deaths.”

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http://bit.ly/WHO_Ecolihttp://bit.ly/WHO_Updateshttp://bit.ly/ECDC_Ecolihttp://www.ncbi.nlm.nih.gov/pubmed/21793740http://www.ncbi.nlm.nih.gov/pubmed/21696328

Links & Resources

in which I was interviewed eighteen times to explain the biology of commensal and pathogenic E. coli for radio and TV sta-tions, and having written two technical notes for EL PAÍS myself and posted one entry into a microbiology blog on this top-ic, I felt satisfied.During the crisis, I perceived the need to have access to channels conveying reliable scientific information at the same speed at which news reached the journalists. I did not err much in my opinions, but I had to use my full knowledge and good judgement to correctly interpret and react to the news as they were conveyed, almost in real time, by the media. Quick access to sound scientific facts will benefit all of us, communicators, governments, citizens, as well as scientists.

What measures can we apply to be safe from EHEC?

HK: The simplest and most efficient way to avoid EHEC infections is strict adher-ence to basic hygiene rules that prevent transmission of the pathogen to humans. These include careful hand washing after using the toilet, to avoid contact with ani-mals and patients who shed EHEC in their stools, avoiding consumption of all kinds of food that can be contaminated with EHEC either primarily or during production, mainly undercooked meat, non-pasteurized milk, and drinking water from unknown sources. Unfortunately, we do not know how to make fresh fruits and vegetables perfectly safe without cooking or radiation – washing these foods, which are, of course very good for human health, is an imper-fect way to get rid of these pathogens.

MV: The speed involved in the exact iden-tification of this bacterial isolate, down to the full sequencing of its genome, has been almost miraculous. On the contrary, discovering where the active focus causing an infective outbreak resides, appears to me as the most difficult question to address. It does not only involve knowledge and tech-nique, but also needs a large amount of ingenuity and even good luck. Identifying the primary dissemination source is diffi-cult to the point that in many instances it is

Dr Jeff Cole, EFB“Microbial physiology lies at the heart of biotechnology, and public perception of biotechnology, like all other aspects of sci-ence, is guided by information available in the press. The consequences of the release of inaccurate information about the recent E. coli EHEC outbreak in Germany illus-trated the urgent need for access by the press to expert advice. The European Fed-eration of Biotechnology strongly endorses the formation by FEMS of a European Microbiology Forum (EMF) for which one task should be coordination of expert advice about microbiology across Europe.”

Dr Huub Lelieveld, EFFOST“Cooperation between European scien-tific organisations such as FEMS, EFFoST and EuCheMS, will provide a strong ba-sis for quickly identifying food safety ex-perts when needed to address food safety incidents. In addition, through the com-munication channels of these organisa-tions, sound scientific information can be quickly distributed. Thereby, unfounded statements that cause unnecessary confu-sion may be avoided.”

Other experts on the EHEC outbreak

never determined. In this respect, my guess is that in a world in which food supplies travel along incredible distances and are handled at countless points in their jour-

neys, traceability and safe handling practic-es are some of the issues in which we have lots of room for improvement.

News from the European Academy of Microbiology (EAM)

The EAM has a new president. Dr Philippe Sansonetti of France replaces Dr Jörg Hacker of Germany. Dr Sansonetti is currently professor at the Institut Pasteur in Paris, France. He also heads the Unité de Pathogénie Microbienne Moléculaire at the same institute.

EAM’s past President Dr Hacker is currently President of the German Academy of Natural Scientists Leopoldina - National Academy of Sciences. FEMS conveys its deepest thanks for the excellent leadership of Dr Hacker in mounting this excellent initiative and wishes Dr Sansonetti the best of luck in promoting EAM towards new frontiers.

The EAM will arrange a meeting on the EHEC 0104:H outbreak at Institut Pasteur in Paris on November 29-30, 2011. Keynote speaker will be Brett Finlay, University of Vancouver, Canada. More information on the EAM can be found on the EAM website: www.europeanacademyofmicrobiology.org.

Dr Philippe Sansonetti replaces Dr Jörg Hacker as EAM President.

Rasko D et al. “Origins of the E. coli Strain Causing an Out-break of Hemolytic–Uremic Syndrome in Germany” N Engl J Med 365;8, 2011 - findings based on genome sequencing suggest that horizontal genetic exchange allowed for the emergence of the highly virulent Shiga-toxin–producing enteroaggregative E. coli O104:H4 strain that caused the German outbreak.Frank C et al. “Epidemic Profile of Shiga-Toxin–Producing Escherichia coli O104:H4 Outbreak in Germany.” N Engl J Med 10.1056/nejmoa1106483, 2011

Important information on the current EHEC outbreak published on July 22 in

New England Journal of Medicine: