Bacterial infections and antimicrobial resistance

Aim and focus

Infections due to antibiotic resistant bacteria are increasing in importance, both in hospitals and in the community. In recent years, drug-resistant strains of the Gram-positive Clostridium difficile emerged worldwide and were identified as causative agents of enteric infections with high mortality and morbidity. Infections due to Gram-negative bacteria that are resistant to first-line therapeutic agents as well as to second-line agents are also more frequent. Early diagnosis and a better understanding of the virulence characteristics of these bacteria should result in the development of more appropriate therapeutic interventions. Additionally, prevention of spread of C. difficile and multidrug-resistant Gram-negatives are important issues in hospital infection control. A healthy and diverse intestinal microbiota is considered an important defence against colonization with C. difficile and multidrug-resistant Gram-negatives. Faecal microbiota transplantation (FMT) is accepted as a standard treatment for recurrent C. difficile infections and is explored for eradication of multidrug-resistant bacteria from the intestinal tract. Other diseases that involve disbalanced microbiota in the intestinal tract, e.g. inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), are also considered for FMT treatment and studies are set up to investigate the precise contents and the active constituents of the donor faeces.

Position in international context

The LUMC Experimental Bacteriology (ExpBac) group closely collaborates with the Dutch Centre for Infectious Disease Control (CIb), the European Centre for Disease Prevention and Control (ECDC) and the United States Centers for Disease Control and Prevention (CDC). The group’s extensive network consists of epidemiologists, microbiologists, geneticists, molecular biologists, and biochemists. Experimental Bacteriology plays a prominent role in the European Study Group on C. difficile under auspices of the European Society for Clinical Microbiology and Infectious Disease (ESCMID) and coordinates numerous activities related to this subject. The group was involved in a number of EU-funded projects concerning C. difficile and these have led to productive collaborations with several leading international institutes, including the Wellcome Trust Sanger Institute (Hinxton, UK), on topics such as single nucleotide polymorphism typing and culturomics. Since 2015, the Department of Medical Microbiology hosts the National Donor Feces Bank (NDFB, www.ndfb.nl), a collaboration between LUMC, VUMC, AMC, HMC, Havenziekenhuis Rotterdam and Wageningen University, supported by ZonMW. The NDFB distributes carefully selected donor material for FMT  of patients with multiple  recurrent C. difficile infections and investigates transmission of protective bacteria and their functional networks. In 2016 the Netherlands Centre for One Health (NCOH, www.ncoh.nl) was founded and the LUMC is one of the partners. The NCOH “aims for an integrated One Health approach to tackle the global risk of infectious diseases”. ExpBac participates in the antimicrobial resistance (AMR) and emerging infectious diseases (EID) themes.

Content / highlights / achievements

Our research group is involved in national and European surveillance studies on the emergence of multidrug-resistant bacteria and C. difficile variants associated with increased virulence. New molecular typing techniques (including next-generation sequencing and proteomics techniques) have been applied to C. difficile and multidrug-resistant Gram-negative bacteria with the aim to understand the occurrence, rise, and spread of these microorganisms. In the area of C. difficile research, the LUMC team coordinated and participated in EU-funded projects (in both FP6 and FP7) on the pathogenesis, epidemiology, and molecular typing of this emerging human pathogen. We participate in an innovative medicines initiative (IMI) on the burden of C. difficile infections in Europe and will lead an European consortium for CDI surveillance supported by the ECDC. We have extensively studied toxin production in C. difficile. In addition, several regulators thought to be involved in DNA replication, virulence, and sporulation have been biochemically characterized and are studied as new targets for antimicrobial agents. The group has expanded its scope to include next-generation sequencing for phylogeny and typing, the investigation of mobile genetic elements, biochemistry,  and collaboratively X-ray crystallography and high-end mass spectrometry. Analysis of the secretome of C. difficile has resulted in the discovery of a highly expressed secreted protease that can be used as a target for intervention, as well as a marker for infection. These research topics have been supported by several grants from ZonMW (prof. dr. E.J. Kuijper, dr. J. Corver) and a Gisela Thier Fellowship (dr. W.K. Smits). Dr. Smits has also secured a Veni and a Vidi grant as part of the Innovation Scheme of the Netherlands Organisation for Scientific Research (NWO). This has established the group as a leading laboratory that uses a multidisciplinary approach towards combating C. difficile infections.
After the first large outbreaks of C. difficile in The Netherlands, the National Reference Laboratory for C. difficile was established, based in the LUMC and financed by CIb) of the Dutch National Institute for Public Health and the Environment (RIVM). In the Reference Laboratory, C. difficile isolates from all over the Netherlands are characterized and related to clinical and epidemiological patient data. Since 2009, the Reference Laboratory also performs a sentinel surveillance with 20 participating hospitals. Together, the activities of the Reference Laboratory and our European activities since 2004 have created a collection of more than 6,000 well-characterized and well-documented C. difficile isolates from various countries. This database is crucial for the analysis of the clinical and epidemiological significance of newly emerging types. It was also used to identify specific DNA markers of epidemic C. difficile strains. Moreover, the collection has been used for validation of lead therapeutics on a contract research basis. In close collaboration with reference laboratories from the UK (University of Leeds) and USA (CDC), commonly used typing techniques as well as promising new techniques have been standardized using a reference set of type strains that is available for distribution worldwide.
Experimental Bacteriology is actively investigating the contribution of gut microbiota to health and disease. 16S microbiome analyses are used, for instance, to study the bacterial complexity in the gut of patients suffering from Graft-versus-Host disease (GVHD), in a collaborative project with the LUMC Department of Paediatrics. Studies have also been designed to investigate association of the microbiota with neuropsychiatric disease. Upon founding the NDFB, the group has also started a new research line on culturomics, which aims to identify and study the bacterial (and possibly viral) composition of complex biological mixtures, such as faeces, through culturing of as many species as possible, including anaerobic and spore-forming bacteria. In combination with next-generation sequencing, it provides a powerful and necessary approach to identify species that are crucial for the protective properties that such complex mixtures may have, for instance against C. difficile infections or multidrug-resistant Gram-negative bacteria. These activities have led to collaborations with and funding from industrial research partners.

Future themes

• Studies of the human gastro-intestinal microbiota including culturomics, and development of intervention techniques to influence its composition.
• Application and implementation of metagenomics and proteomics for the recognition of multidrug-resistant bacteria and fungi to understand their epidemiology.
• The use of comparative genomics in the identification of virulence traits in epidemic strains of C. difficile.
• Establishing the role of differential responses of C. difficile strains to environmental and host-induced stress factors, such as nutrient availability and exposure to antimicrobials.
• Establishing which proteins are involved in DNA replication and genomic stability in C. difficile, and determining how the regulation of this process differs from that in closely related bacteria; exploring the possibilities of targeting DNA replication for development of new antimicrobial agents with strong species specificity.

Cohesion within LUMC

Infectious diseases, bioinformatics, and biomolecular interaction studies are major LUMC research themes, which provide this project with several internal collaborations. Most important are the interactions within the LUMC Medical Research Profile “Immunity, Infection and Tolerance”, with the Department of Infectious Diseases, the Leiden Genome Technology Center (LGTC) and the Center for Proteomics and Metabolomics (CPM). With the latter, there is an excellent synergy with respect to the proteomic and genomic characterization of bacteria to study resistance and virulence markers. This has resulted in a successful ZonMW grant application entitled “A next-generation mass spectrometry platform for rapid and accurate identification of multidrug-resistant Gram-negative bacteria”. Whole genome sequencing and SNP typing as well as (future) microbiota studies are performed in collaboration with the Sequencing Analysis Support Core (SASC) in the LUMC, the company GenomeScan and with CPM to study microbiota metabolomics. In our own Department, the interactions with the Molecular Virology research group are highly relevant with regard to extensive expertise in molecular biology and host-pathogen interactions. The recently started initiatives on studies of the microbiota are performed with partners from the LUMC Departments of Parasitology, Infectious Diseases, Pathology, Chemical Immunology, Neurology, Pulmonary Diseases, Paediatrics and Hematology and cross the borders of several research profile areas within LUMC.