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Microbiologists fight drug-resistant pathogens

Ignasi Roca, Elisabet Guiral and Jordi Vila  
Department of clinical microbiology, CDB, Hospital Clinic, IDIBAPS, Barcelona, Spain
TROCAR partners

The health care systems of most European countries are based on a continuum from acute care hospitals to other health care facilities and the community.

Such a framework provides the perfect opportunity for the rapid dissemination of high-risk, resistant clones (HiRiC) or genetic-resistant bacteria elements.

The emergence of multidrug resistant clones is now becoming a serious healthcare issue and resistance continues to spread not only within the nosocomial setting but also in community-
acquired organisms.

We are in desperate need for appropriate control measures as well as rapid identification procedures for isolates of a particular epidemicity.

The TROCAR project (Translational Research on Combating Antimicrobial Resistance), co-ordinated by Dr Jordi Vila at the Hospital Clínic de Barcelona, was created in 2009 as a consortium of up to 15 partners from 10 different European countries in an attempt to provide specific answers to three main concerns:

Are certain resistant strains more epidemic than others? Are certain strains more prone to persist in the human environment? If so, why?

Do epidemic and persistent strains have specific virulence, physiological, colonisation or transmission-facilitating traits that non-epidemic strains do not have?

What is the origin and mechanisms of acquisition of these fitness-increasing traits in resistant bacteria? Might the elucidation of these mechanisms provide new insights for prediction and intervention?

The target pathogens
The driving concept of TROCAR is to investigate the fundamentals and the epidemiology of new highly virulent multiresistant strains of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococcus (VRE) species, extended-spectrum, metallo- and acquired AmpC beta-lactamase-producing (ESMAC BLs) enterobacteriaceae, and multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii.

In this regard, the TROCAR project was meant to focus on three major strategic aims:

A definition for the major high-risk resistant clones circulating in Europe
The promotion of collaborative European research to investigate specific traits associated with virulence, transmission, persistence and resistance of epidemic clones in comparison with non-epidemic clones
The development of bio-informatic tools to fully exploit the genomics data and allow the rapid identification of resistant strains with heightened epidemic potential.

To fulfil these aims, the TROCAR partners have been organised into eight different work-packages covering the different relevant pathogens, the exchange and monitoring of data and dissemination of results.

Halfway through the project, the consortium has achieved several important milestones that will contribute towards completing the objectives of the original proposal. In this sense, the TROCAR members have agreed on the following definition of ‘high-risk clones’ (HiRiC):

“High-risk, resistant clones are those bacterial clones that associate:

“Mechanisms of resistance to antibiotics of critical clinical importance and 2a) the ability to be transmitted with high efficiency among hospitalised patients or 2b) with particular ability to produce severe or invasive infections; or 2c) the ability to efficiently colonize human hosts during long periods of time.”

A catologue of ‘high-risk’ strains
The combined effort of the consortium members has resulted in the establishment of an inventory of strains for MRSA, VRE, A. baumannii, P. aeruginosa and ESMAC-BLs-producing enterobacteriaceae according to their epidemiological impact and multidrug-resistant profiles. The addition of strains is expected to go on throughout the length of the project.

These catalogues, together with the epidemiological information provided by the partners, have been used to draw a map representing outbreaks of VRE from various hospitals in France, Germany and Finland, allowing the identification of the major VRE clones causing outbreaks in three participating European countries.

Similarly, the geographical abundance of S. aureus/MRSA clones has been displayed on a web-based mapping tool using a Google interface (publicly available at, and a multi-locus sequence typing (MLST) database for S. aureus/MRSA has also been established. Together they will allow TROCAR partners to concentrate on population analyses based on both MLST and next-gen sequencing data.

The phylogeny of epidemic MRSA, which is widely disseminated in central Europe and attributed to clonal lineages ST22 and ST225, was reconstructed by means of genome-wide single nucleotide polymorphism analysis, which has allowed the identification of epidemic subpopulations in clonal complexes and has also shown that community-acquired MRSA from both clonal lineages represent separate subpopulations. We are also currently focusing on the development of summary statistics to describe geographic structuring on a continental scale.

The origin of resistance
The dynamics and persistence of E. faecium clones from bacteriemic patients have been studied as a model of the evolution of HiRiC, revealing dominance and persistence of related CC17 sequence types (STs). These STs, enriched in IS16 element and virulence/epidemicity markers (i.e. esp and hyl genes) might contribute to hospital adaptation and might have supplied a substrate for the emergence of vancomycin resistance. Moreover, the capacity of spread of a new type of vancomycin resistance in E. faecium is under evaluation.

On investigating the epidemiology of A. baumannii, TROCAR members have demonstrated the worldwide dissemination of OXA-23-producing strains. The resistome of A. baummannii is under investigation and the mechanisms of resistance to tigecycline and colistin have also been studied. The overexpression of the efflux pump AdeABC was associated with resistance to tigecycline, whereas resistance to colistin was related to changes in the lipopolysaccharide (LPS) and in the expression of some outer membrane proteins.

The TROCAR consortium has shown that biofilm formation increases the survival rate of A. baumannii on dry surfaces and may contribute to its persistence in the hospital setting, increasing the probability to cause nosocomial infections and outbreaks.

The TROCAR project has also contributed to the clarification of carbapenem resistance in isogenic carbapenem-susceptible/resistant A. baumannii. All potential mechanisms of resistance to carbapenems – such as carbapenemase production, changes in outer membrane proteins, efflux pumps overexpression and changes in penicillin-binding proteins (PBPs) – have been investigated, finding that a normally high level of carbapenem resistance is associated with the combination of several mechanisms.

Extensive research on problem Enterobacteriaceae
The collection of ESMAC-BLs-producing Enterobacteriaceae contains clinical isolates from Spain, France, UK, Greece and Italy, and it is also being investigated to identify the type of beta-lactamases circulating in Europe. A pattern of sudden increases in the prevalence of isolates with particular extended-spectrum beta-lactamse (ESBL) types (especially of CTX-M family enzymes in the community) has been recognised in several European countries.

Different studies performed by TROCAR 
Partners have shown Enterobacteriaceae producing Verona integron-encoded metallo-beta-
lactamase (VIM) and Klebsiella pneumoniae carbapenemase (KPC) in different countries, 
however, special relevance should be given to the E. coli that produces New Dehli metallo-beta-
lactamase (NDM) and K. pneumoniae isolated in the UK.

Most of these genes are plasmid mediated, and different plasmids such as those carrying CTX-M-14, TEM-24. VIM-1 and CTX-M-3 are being currently characterised. Moreover, plasmids carrying CTX-M-1, OXA-48 and SHV-12 have been selected for full sequence analysis.

The inventory of strains has also allowed the selection of five MRSA, two VRE, four P. aeruginosa and three Acinetobacter specific clones that fulfil the HiRiC criteria defined by the consortium. The genomes of these HiRiC clones are now being sequenced and will be compared with those of non-virulent counterparts to investigate candidate genetic elements involved in enhanced virulence and epidemicity.

Establishing the network
During this first year and a half of TROCAR development, two workshops have been organised in Bilthoven, Netherlands.

The purpose of these workshops was to bring representatives from national laboratories in contact with scientists from the TROCAR consortium to establish a collaborative TROCAR network concerning surveillance of S. aureus and ESMAC-BLs-producing enterobacteriaceae in Europe.

Part of the data produced during the first 
part of the project has already been published and it can be found in the TROCAR website ( The success of this proposal is the generation of large volumes of nucleotide sequence data from isolates of major bacterial 
disease agents that together represent an extreme public-health burden throughout Europe. These data will provide powerful insights into the genetic basis of the success of these lineages as highly transmissible, virulent and/or resistant pathogens.

Therefore, the project is linked to the data generated from the full genome sequence of the strains that we have selected. Full genome sequences will help to pinpoint the genes or genetic islands responsible for heightened virulence 
and/or resistance, and the characterisation of novel genes will lead to new targets for inter-

This project is also focused on the characterisation of mobile elements within the populations, particularly plasmids, as these elements often carry the genes responsible for drug resistance and their role in virulence is largely unclear.

As for the chromosomal data, data for mobile elements will be generated both on population and genomic scales, providing novel and powerful perspectives for examining their evolution and dissemination.

By combining the outputs of the project, it will be possible to provide tools for monitoring the spread of key community and nosocomial pathogens, to provide the scientific basis for an early warning system when isolates of a particular epidemicity appear in the community and nosocomial settings.

We will also be able to:

Create background knowledge for combating epidemicity and virulence
Characterise specific genetic elements carrying genes encoding ESCMAC-BL
Identify potential reservoirs of antibiotic resistance genes among community and nosocomial pathogens involving multi-drug resistance
Establish the bioinformatic tools and infrastructure necessary to achieve the ultimate TROCAR goal, which is “to recommend novel control measures to limit or prevent the spread of highly virulent 
multi-drug resistant clones”.

TROCAR Partners:
Instituto de Tecnologia Química e Biológica (ITQB), Oeiras, Portugal (H. de Lencastre); Laboratoire de Microbiologie, Université de Caen Basse-Normandie, Caen, France; National school of Public Health, Athens, Greece; National Public Health Institute, Helsinki, Finland; Department of Molecular Biology, University of Siena, Siena, Italy; University Medical Center Groningen, Groningen, Netherlands; European Society of Clinical Microbiology and Infectious Diseases (ESCMID); Department of Bacteriology-Virology, Hospital Bicêtre, Paris, France; Department of Biology and Biochemistry, University of Bath, United Kingdom; Robert Koch Institute, Wernigerode Branch, Germany; Health Protection Agency, London, United Kingdom; Servicio Madrileño de Salud, Servicio de Microbiología, Hospital Ramón y 
Cajal, Madrid, Spain; Sistemas Genómicos, S.L., Paterna, Spain.