Researchers from the Institut Pasteur, Inserm, CNRS and Paris Descartes – Sorbonne Paris Cité University recently published a large-scale study in Nature Genetics based on almost 7,000 strains of Listeria monocytogenes — the bacterium responsible for human listeriosis, a severe foodborne infection. Through the integrative analysis of epidemiological, clinical and microbiological data, the researchers have revealed the highly diverse pathogenicity of isolates belonging to this bacterial species.

Comparative genomics led them to discover new virulence factors, which were demonstrated experimentally as involved in cerebral and fetal-placental listeriosis. In addition, this research points to the importance of using new reference strains, which are representative of the hypervirulent lineages identified here, for experimental research on Listeria monocytogenes pathogenesis.

Tissue infected by Listeria (bacteria appear in red). YH Tsai, M Lecuit, © Institut Pasteur

In France, as in many other countries, the Listeria monocytogenes bacterium — which is responsible for listeriosis, a severe foodborne infection, especially in pregnant women and the elderly — is subjected to a stringent microbiological monitoring system, coordinated at the Institut Pasteur by the National Reference Center (CNR) for Listeria together with the French Institute for Public Health Surveillance (InVS). Researchers from the Biology of Infection Unit (Institut Pasteur/Inserm), headed by Marc Lecuit (Paris Descartes – Sorbonne Paris Cité University, Necker-Enfants Malades Hospital, AP-HP) and home to the Listeria CNR, together with the group led by Sylvain Brisse in the Microbial Evolutionary Genomics Unit (Institut Pasteur/CNRS), recently published the findings of a broad study of close to 7,000 strains of Listeria monocytogenes isolates collected over the past nine years for monitoring purposes.

First and foremost, bacterial molecular genotyping revealed considerable heterogeneity within the L. monocytogenes species, and showed that strains can be categorized into distinct genetic families (or clonal groups). By analyzing epidemiological data, the researchers showed that some of these clonal groups are more frequently associated with human infections, while others are closely linked to food. The comprehensive analysis of detailed clinical data from over 800 patients with listeriosis showed that the strains most often associated with infections are more frequently isolated in the least immunodeficient patients while the strains most commonly linked to food mainly infect the most immunodeficient individuals. In addition, the strains most often involved in infections appear to be the most invasive as they infect the central nervous system and fetus more often than the strains most commonly associated with food. These findings suggested the existence of hypervirulent strains — an hypothesis that scientists confirmed thanks to a humanized mouse model of listeriosis they developed previously[1].

To uncover the genetic basis of this hypervirulence, the researchers sequenced the genomes of around a hundred strains that are representative of the most prevalent clonal groups. Comparative analysis of these genome sequences identified a large number of genes closely linked to the hypervirulent clonal groups, including one which was demonstrated as involved in the cerebral and fetal-placental tropism of L. monocytogenes experimentally.

These results pave the way to a detailed understanding of the mechanisms underlying L. monocytogenes central nervous system and fetal-placental invasion.

While current research on L. monocytogenes is conducted with so called “reference” strains, which are not hypervirulent, this research supports the use of hypervirulent strains representative of human infections, to ensure the clinical and pathophysiological relevance of experimental research.

This study illustrates the exceptional power of harnessing the biodiversity of a given microbial species (here L. monocytogenes) and integrating epidemiological, clinical, bacteriological and experimental data to study the biology of infection in a clinically relevant manner.

[1] See the press release (September 17, 2008)

Using innovative technology, scientists from the Institut Pasteur and Inserm have filmed in vivo the process by which an AIDS vaccine candidate, developed by the French Vaccine Research Institute and the ANRS, triggers the immune response. This previously unseen footage clearly shows how the vaccine recruits the immune cells needed to destroy infected cells. These results, published in the journal Nature Medicine on December 21, 2015, shed new light on the mode of action and potential of this vaccine.

The aim of the study conducted by scientists from the Dynamics of Immune Responses Unit (Institut Pasteur / Inserm / VRI), directed by Inserm research director Philippe Bousso, was to observe the effect of the HIV/AIDS vaccine candidate MVA-HIV – currently undergoing clinical trials by the French Vaccine Research Institute (VRI) and the ANRS – on the immune response.

The scientists administered the vaccine to healthy mice, then observed in real time how cells from the immune system were mobilized to the lymph node, the organ where the vaccine response is developed, in just a few hours.

For the first time, using a powerful, non-invasive microscopic imaging technique, the scientists were able to watch in vivo and in real time as the vaccine induced the formation of the inflammasome, a complex assembly of proteins with a highly specific structure which appears in macrophages, the first immune cells targeted by the vaccine.

The inflammasome promotes the maturation of the chemical messenger interleukin (IL)-1 but also induces macrophage death, thereby releasing this inflammatory messenger in the lymph node. This signal triggers a chain reaction which assembles several key players of the immune system in the lymph node, including killer cells, which are vital for the vaccine response.

These in vivo films have given the scientists a detailed picture of the main stages in the mechanistic action of this vaccine and highlighted an important pathway that orchestrates the effective mobilization of the immune response.

“This is the first time that the formation of this original structure, the inflammasome, has been observed in vivo and in real time,” commented Philippe Bousso. “Our research demonstrates the potential of the vaccine candidate MVA-HIV to trigger a significant, diverse immune response.”

This research was supported by the French Vaccine Research Institute, the French Foundation for Medical Research and the European Research Council (ERC).

Image/ film: In green, the cells targeted by the MVA-HIV vaccine soon activate the inflammasome (circled green spots) and die. This signal triggers a mass mobilization of immune cells. © Initut Pasteur

Éric Vivier’s team at Marseille-Luminy Immunology Center (CIML), a CNRS, Inserm and Aix-Marseille University research centre, in collaboration with Gabrielle Belz’s team at Walter and Elisa Hall Institute (WEHI), Melbourne, sheds new light on the dynamics of the immune networks that protect our intestine. During episodes of bacterial diarrhoea, innate lymphoid cells (ILCs) “share the work” with memory T lymphocytes, but can also compensate for them in the event of failure.
The authors also show that ILCs protect the appendix from potential damage caused by infection, a finding that may confer a new status on this organ. These discoveries are presented this Monday, 30 November, in the scientific journal Nature Immunology.

The intestinal immune system includes different populations of cells from the innate and adaptive immune system that protect us from infection. Once the pathogen is detected, cells from the innate immune system launch the initial attack, and call for reinforcements from the B and T lymphocytes of the adaptive immune system in order to “finish the work” and memorise the intruder’s profile. Although the sequence of events is known, cooperation between the different components is still poorly understood. The researchers are thus trying to uncover the relationships that form within the intestine between the T lymphocytes and the innate lymphoid cells (ILCs). Discovered simultaneously in 2008 by 12 laboratories, including that of Éric Vivier, ILCs represent a new type of lymphocyte that had been completely unknown until then. These cells can be likened to a “rapid version” of T lymphocytes. Divided into different groups, they have the morphology of a lymphoid cell, and produce the same cocktails of cytokines as the T lymphocytes, but they uniformly lack antigen-specific receptors.

The authors are now revealing another aspect of their biology: their choreography with the T lymphocytes. Using the bacterial species Citrobacter rodentium in the mouse as a model of human diarrhoea caused by enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC), they have thus demonstrated that the protective action of T lymphocytes and of an ILC subtype (ILC3 NCR+) is not only redundant, but that the latter can even “take over the work” where the T lymphocytes fail.

“This is the first time that redundancy between these 2 cell populations has been formally demonstrated,” says Éric Vivier. “All the evidence suggests that these two defence systems were co-selected during evolution to ensure optimal protection against infection.”

Ultimately, this discovery could thus lead to new therapeutic approaches to intestinal diarrhoea, a public health challenge of planetary proportions, since every year nearly 4 billion people are affected by these diseases, which represent nearly 4% of deaths worldwide.
Furthermore, this study includes another unexpected discovery: during infection, these same ILCs protect the caecum/appendix pair from inflammation and potential injury.

“The caecum and appendix have long been considered vestigial organs in terms of immunity. Our work is part of a quite different hypothesis, which proposes a role for this part of the intestine as a refuge for commensal bacteria during infection. Thus the ILCs would help to nurture the “good” bacteria that help to maintain the balance of the intestinal microbiota and hence fight infections more effectively,” concludes Professor Éric Vivier.

Announced at the end of 2014 during the final phase of research and development, the rapid diagnostic test for Ebola virus disease developed by the French Atomic Energy Commission (CEA), with support from the Inserm Jean Mérieux P4 high-level biological containment laboratory (Lyon), has been assessed in the field, including in Guinea, over a period of several months. This assessment now allows the test to be CE marked, leading to its authorisation for use as a diagnostic tool. The eZYSCREEN^® test will thus help to enhance the available means of control, particularly by detecting residual sporadic cases among symptomatic patients, or by post-mortem identification.

The rapid diagnostic test for Ebola, developed in the public health emergency of 2014 and 2015 by a team from the Life Sciences Division (DSV) of CEA, with support from the Inserm Jean Mérieux P4 high-level biological containment laboratory (Lyon), allows diagnosis of the disease within 15 minutes, using a few drops of blood or serum. An agreement has been drawn up with the Vedalab company (Alençon), which manufactures it.

In contrast to laboratory tests, this easy to use field test does not require sophisticated instrumentation or even electricity, and can be used by non-specialist staff. The result is read directly and visually. Storage for eight and a half months at 30°C, and for 14 days at 45°C, did not affect its performance, proving its high robustness and stability.

The eZYSCREEN^® test was assessed under real conditions, including in the Ebola Treatment Centres in Coyah and Forécariah in Guinea (French Red Cross), and part of the analyses were also conducted in Donka National Hospital (Conakry, Guinea). These studies ended in August 2015. 

The rapid test shows excellent specificity, eliminating almost any risk of false positive results, and an appropriate degree of sensitivity for its intended use, i.e. to very rapidly identify the maximum number of cases possible in situ with a view to preventing epidemics.

These results enabled CE marking of the eZYSCREEN^® test for Ebola, which is essential for its use during the sporadic cases that continue to occur in Guinea and Sierra Leone. The objective is to be able to avoid the start of a new large-scale epidemic outbreak.

Discussions are underway between CEA and diagnostics industries in order to allow the widest possible distribution of the test.

For this body of work, the Technological Innovations for Detection and Diagnosis (Li2D/DSV) Laboratory in the CEA Marcoule Centre has just received the Societal Resilience Trophies Technological Innovation Prize for 2015, awarded by the French High Committee for Civil Defence.

The efforts of the international community for the past ten years in the fight against malaria have reduced the number of disease-related deaths. The emerging resistance to standard therapies threatening South-East Asia, and new research carried out by the team led by Françoise Benoit-Vical, Inserm Research Director in the CNRS (French National Centre for Scientific Research) Coordination Chemistry Laboratory in collaboration with the Pasteur Institute and Inserm, are not reassuring factors. The in vitro examination of a strain of parasites solely exposed to artemisinin (the base compound for standard therapy) demonstrates the development of a widespread resistance to most other anti-malarial drugs. This new resistance cannot be detected by tests currently used and represents an additional threat to antimalarial treatments in the field.This research is published in the journal Emerging Infectious Diseases.

Malaria is caused by a parasite propagated through bites from infected mosquitoes from the Anopheles genus. This disease is most prevalent in tropical areas and still responsible for over 600,000 deaths each year. Policies to combat this disease have led to a 60% decrease in mortality over 15 years. However, artemisinin, the pharmaceutical base compound for antimalarial therapies, is encountering increasing clinical failure due to emerging resistance throughout South-East Asia. As of yet, this resistance has not been observed on the African continent.

Artemisinin is an active ingredient from a Chinese plant whose benefits have been known for over 2,000 years. It is used in combination with other antimalarial agents. The value of these combination therapies lies in the assurance that even if the parasite develops a resistance to one molecule, it is less likely to develop a resistance to both molecules.

Scientists must nevertheless stay one step ahead of the parasite, given the recent and rapid development of resistance to artemisinin. It is against this background that the team led by Françoise Benoit-Vical, Inserm Research Director in the CNRS Coordination Chemistry Laboratory in Toulouse, in collaboration with Inserm in Toulouse and the Pasteur Institute in Paris, is studying the resistance mechanisms developed by Plasmodium falciparum, the parasite responsible for malaria, and researching new antimalarial drugs.

Researchers have shown that parasites who survive in vitro in the presence of only artemisinin for five years develop a widespread resistance to most other artemisinin-based or non-artemisinin-based antimalarial therapies, including partner molecules present in combination therapies used in endemic areas.

Scientists have demonstrated that these parasites do not exhibit any known mutation in resistance genes. However, they circumvent the toxic effect of the drugs by a phenomenon known as quiescence. Parasites are capable of suspending their development during exposure to antimalarial agents. As soon as they are no longer subjected to antimalarial therapy, the parasites “wake-up” and proliferate once again.

The new multi-drug resistance based on this quiescence phenomenon cannot be detected by tests currently carried out to analyse parasitic resistance. “In vitro tests carried out using the patient’s blood predict high sensitivity and, therefore, the treatment’s effectiveness, while parasites are resistant because they are quiescent. As such, it is essential to conduct research with relevant and appropriate tests in the field if the multi-resistant phenomenon that we identified in vitro is also present, in order to design therapeutic strategies accordingly.” explains Françoise Benoit-Vical.

Our bodies have ten times the amount of microbes than human cells. This set of bacteria is called microbiota. In some instances, bacteria known as pathogens can cause infectious diseases. However, these micro-organisms can also protect us from certain diseases. Researchers from Inserm, Paris Descartes University and the CNRS (French National Centre for Scientific Research), through collaboration with teams from China and Sweden, have recently shown how microbiota protects against the development of type 1 diabetes. This research is published in the Immunity journal, 4 August 2015.

A pancreatic islet of Langerhans expressing the immunoregulator antimicrobial peptide CRAM (in red). The insulin-producting beta-cells are in green and the glucagon-producting alpha-cells are in blue. © Julien Diana

To combat pathogens, the immune system has developed various mechanisms to detect, defend against and even destroy micro-organisms that are harmful to the body. This includes antimicrobial peptides and natural proteins that destroy pathogenic bacteria by disrupting their cellular membrane. Not only are they produced by immune cells, they are also produced by cells whose functions are not immune-related.

A research team coordinated by Julien Diana, an Inserm Research Fellow at Inserm Unit 1151 “Institut Necker-Enfant Malades” [Necker Institute for Sick Children] (Inserm/CNRS/Université Paris Descartes), is focussing on a category of antimicrobial peptides, i.e. cathelicidins. Apart from their protective function, these peptides have also exhibited immunoregulatory abilities against several autoimmune diseases. As such, scientists hypothesise that cathelicidins may be involved in the control of type 1 diabetes, an autoimmune disease where certain cells in the immune system attack beta cells in the pancreas which secrete insulin.

Firstly, they observed that beta pancreatic cells in non-diseased mice produce cathelicidins and that, interestingly, this production is impaired in diabetic mice.

To test this hypothesis, they are injecting diabetic mice with cathelicidins where production is defective.

“Injecting cathelicidins inhibits the development of pancreatic inflammation and, as such, suppresses the development of autoimmune disease in these mice” states Julien Diana.

Given that the production of cathelicidins is controlled by short-chain fatty acids produced by gut bacteria, Julien Diana’s team are studying the possibility that this may by the cause of the cathelicidin deficiency associated with diabetes. Indeed, researchers have observed that diabetic mice have a lower level of short-chain fatty acids than that found in healthy mice.

By transferring part of the gut bacteria from healthy mice to diabetic mice, they are re-establishing a normal level of cathelicidin. Meanwhile, the transfer of micro-organisms reduces the occurrence of diabetes.

For the authors, “this research is further evidence of the undeniable role microbiota plays in autoimmune diseases, particularly in controlling the development of autoimmune diabetes”. 

A large-scale collaborative study, conducted by a French team comprising of the Infectious and Tropical Disease departments of the AP-HP, the hospital bacteriology and hygiene laboratories, the Research Unit of the Bichat – Claude Bernard hospital, the Paris-Diderot University, Inserm, the Pasteur institute and InVS, revealed the acquisition of multi-resistant enterobacteria (MRE) in one in two travellers returning from a stay in a country situated in a tropical region. These results were published in the international journal Clinical Infectious Diseases on 22^nd April, 2015.

©Fotolia

Enterobacteria are microorganisms naturally present in our digestive tract.  While some are completely harmless, others can be responsible for serious infections. In such cases, the patients are treated with antibiotics. Unfortunately, some of these bacteria are resistant, making treatment of these diseases difficult.

The teams coordinated by Sophie Matheron and Etienne Ruppé carried out a study on 824 healthy people before and after a stay in a tropical region (sub-Saharan Africa, Asia, or South America). These people were asked to answer a medical questionnaire and to provide a stool sample in the week before their departure and within three days of their return. This step was repeated for travellers carrying multi-resistant enterobacteria (MRE) until the samples were negative.

The study revealed an overall acquisition frequency of MRE in the digestive tract during the trip of 51%, i.e. one in two travellers. This increased frequency differs depending on the geographical regions visited: on returning from Asia, over 72% of travellers were carriers, more than 47% of those returning from sub-Saharan Africa were carriers, and a little more than 31% of those returning from South America were carriers.

The acquisition of these microorganisms is favoured by the taking of antibiotics (which alters the natural “barrier” protection effect of the bacteria normally present in the digestive tract), the occurrence of diarrhoea during the trip, and the type of trip.  The open stays (organised trips, the family or the backpacker type) present a greater risk than closed stays in hotel-clubs. The prolonged follow-up showed that 95% of the travellers spontaneously eliminate the MRE in the three months after their return.

This study reminds us of the importance of the prudent use of antibiotics (often consumed in an excessive way in cases of simple diarrhoea) and the importance of respecting the preventative and basic hygiene rules during the trip (wash food, wash hands, drink water from a bottle, etc.). Thanks to dedicated consultations, the international vaccine and advice centres provide this information to travellers. They also give other advice and prescriptions depending on the destination (vaccination, prevention of malaria).

Finally, these results highlight the necessity to study the involvement of MRE in patients reporting an infection in the three months following their return from a tropical region. Thus, the health professionals in towns and hospitals may prescribe a treatment adapted to a possible antibiotic resistance.

It should be noted that travellers carrying MRE are not ill and, on this ground alone, have no reason to consult a doctor.