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EUROPE’S E. COLI CRISIS

Microbes turn wild 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 Investigaciones Biológicas (CIB) at CSIC in Madrid, his first contact with E. coli research was in 1973 during a long-term fellowship.

What is your role in the E. coli research?

Dr Helge Karch (HK): We focus on enterohemorrhagic E. coli (EHEC) which can cause, in addition to diarrhoea, systemic complications, most notably the haemolytic uraemic syndrome (HUS).

Our work addresses several broad, but interacting aspects of human infections: 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 involved 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, colonization factors including the Sfp fimbrial adhesin, and a family of immunoglobulin-binding proteins.

Current understanding strongly suggests that Stxs are the predominant virulence 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-glycosphingolipid 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-

Escherichia coli is a Gram-negative, rodshaped bacterium that is commonly found in the lower intestine of warm-blooded organisms. 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 outbreak. Source: Wikimedia Commons

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 activities focuses on mechanisms involved in evolution of these strains in the environment and during infection. These analyses include functional analysis of pathogen-host interactions as well as systematic and thorough populationand geographic interrogation of microbial molecular epidemiological markers.

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

From the Editorial Team June proved to be a busy month concerning 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 bacterium 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. Various 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

est organisms, manage to regulate so precisely their growth and their proliferation. How they coordinate, both in time and space, the division 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 bacterial cell division cycle has been largely derived

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, nucleoid 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 investigated how the genes that encode a large number of them are grouped in a chromosomal region. This region is conserved in rodshaped bacteria and the expression of its genes is controlled through complex signals that ensure 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, molecular 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 disproportionate frequency of severe neurological complications, such as encephalopathy and epileptic seizures. The reasons for the atypical age distribution 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.

Source: Helge Karch

Source: Inés poveda

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.

Miguel Vicente is Professor of Research at “Centro Nacional de Biotecnología (CNBCSIC)” 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 stability 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, DIVINOCELL, gathering groups from eleven European and South American institutions aiming at discovering new drugs to combat infections caused by Gramnegative bacteria.

EAEC. We hypothesize that the strong adherence phenotype enhances systemic absorption of Stx produced by the colonizing bacteria; this, indeed, could increase the rate and severity of systemic complications (HUS), because 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 How virulent is the HUSEC041 clone that genes that encode nasty proteins, those that is causing the current EHEC epidemic as cause disease in humans and animals. The compared to other EHEC strains and to the EHEC strains cause a severe bloody diarnormal E. coli? rhoea. In plain words, they damage the cells that line our intestine, sitting on their top, HK: The outbreak strain seems to be extraormodifying their membranes and injecting dinarily virulent as indicated by the unpreproteins into their interior that subvert and cedented number of patients that developed deteriorate their HUS and then physiology. The died. The reasons “By the end of July 2011, the enterohaemorrhagic strain, for the apparentEscherichia coli EHEC strain 0104:H yielded more than O104:H4 isolated from the ly increased virulence of the out- 3, 800 confirmed infections and 823 cases of haemolytic patients of this outbreak, posbreak strain is yet uraemic syndrome (HUS), with 44 deaths.” sesses other ununknown and is desirable traits. One is a gene that codes for a the subject of ongoing research in my laboratoxin, called Shiga, that ends up in the bloodtory. As we have demonstrated in our recent stream, acting mostly within the capillaries of publications one reason can be that the strain smallest diameter. The Shiga toxin even alters efficiently combines major virulence characsome components of the immune system and teristics of EHEC and enteroaggregative E. it has a final deleterious effect on the malcoli (EAEC), i.e. production of Stx and abilfunctioning of the filtering devices present in ity to intensively adhere to intestinal epithethe kidneys. This is the reason why, when the lial cells in an aggregative manner typical for

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 classified as STEC, meaning Shiga toxin E. coli.

This strain has also proven to be very effective in attaching to the surface of the intestinal cells, making it more dangerous because the infection stays longer. Consequently, it has also been classified as enteroaggregative

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 cephalosporins and susceptible to carbapenems (ertapenem, imipenem, meropenem). Furthermore, all outbreak isolates analysed in our laboratory are resistant to trimethoprim/ sulfamethoxazole and susceptible to fluoroquinolones (ciprofloxacin) and aminoglycosides (gentamicin, tobramycin).

MV: E. coli is a Gram-negative bacteria, meaning that it has an envelope formed by a rigid layer of peptidoglycan sandwiched in between 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 infections would provide a mixed blessing, as they cause bacterial lysis and consequently the release of free toxin into the patient organism.

Other antibiotics, as quinolones, that interfere 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 resistance against heavy metals. On the positive side, it appeared to be sensitive to carbapenems, a class of beta-lactams used in hospitals, less prone to be destroyed by the betalactamase 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 frequently 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 criminal purposes unlikely. In addition, cooking Colonies of Escherichia coli bacteria grown on a Hektoen enteric (HE) agar plate medium Source: CDC

This map depicts the 2011 E. coli O104:H4 outbreak. Source: Wikimedia Commons

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 developed just after the characterization of the toxins 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 active clinical development, Shigamabs™, chimeric antibodies to Stx1 and Stx2. This program 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 eculizumab 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,

in which I was interviewed eighteen times to explain the biology of commensal and pathogenic E. coli for radio and TV stations, and having written two technical notes for EL PAÍS myself and posted one entry into a microbiology blog on this topic, 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.

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 practices are some of the issues in which we have lots of room for improvement.

Other experts on the EHEC outbreak

What measures can we apply to be safe from EHEC? HK: The simplest and most efficient way to avoid EHEC infections is strict adherence 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 animals 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 imperfect way to get rid of these pathogens.

Dr Jeff Cole, EFB “Microbial physiology lies at the heart of biotechnology, and public perception of biotechnology, like all other aspects of science, 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 illustrated the urgent need for access by the press to expert advice. The European Federation 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.”

News from the European Academy of Microbiology (EAM)

MV: The speed involved in the exact identification 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 technique, but also needs a large amount of ingenuity and even good luck. Identifying the primary dissemination source is difficult to the point that in many instances it is The FEMS Focus is published by the FEMS Central Office Keverling Buismanweg 4 2628 CL Delft The Netherlands Tel: +31-15-269 3920 Fax: +31-15-269 3921 E-mail: fems@fems-microbiology.org FEMS is a registered charity (no. 1072117) and also a company limited by guarantee (no. 3565643). © 2011 Federation of European Microbiological Societies Design: Zak Princic Production: Ilumina.si

Dr Huub Lelieveld, EFFOST “Cooperation between European scientific organisations such as FEMS, EFFoST and EuCheMS, will provide a strong basis for quickly identifying food safety experts when needed to address food safety incidents. In addition, through the communication channels of these organisations, sound scientific information can be quickly distributed. Thereby, unfounded statements that cause unnecessary confusion may be avoided.”

Dr Philippe Sansonetti replaces Dr Jörg Hacker as EAM President.

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.

Important information on the current EHEC outbreak published on July 22 in New England Journal of Medicine: Rasko D et al. “Origins of the E. coli Strain Causing an Outbreak 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

Links & Resources http://bit.ly/WHO_Ecoli http://bit.ly/WHO_Updates http://bit.ly/ECDC_Ecoli http://www.ncbi.nlm.nih.gov/ pubmed/21793740 http://www.ncbi.nlm.nih.gov/ pubmed/21696328