DRESP2 - Role of mobile genetic elements in the spread of antimicrobial drug resistance

EC contribution: : € 1.852.000
Duration: : 36 months
Starting date: : 01/01/2006
Funding scheme: : Specific Targeted Research Project
Keywords: : drug resistance, epidemiology, molecular diagnostics, in vitro diagnostic medical device, mobile genetic element
Contract/Grant agreement number: : LSHM-CT-2005-018705
Project web-site: : http://www.dresp2.com/

Background:

Mobile DNA provides a major contribution to the spread of antimicrobial resistance, by recruiting new resistance genes in bacterial pathogens and facilitating their horizontal spread. While much is known about individual resistance genes and mechanisms, the nature of the mobile elements and their transfer mechanisms have been clarified only in few cases, and very little is known about their molecular epidemiology. A comprehensive understanding of these aspects could provide a significant breakthrough toward combating dissemination of resistance determinants. The DRESP project aims at investigating these aspects, focussing on few major families of genetic elements that carry a number of important and emerging resistance determinants: i) the double resolvase elements (DR-elements) of gram-positive cocci, involved in spread of macrolide resistance genes in streptococci; ii) the related DR-elements in Gram-negative cocci, an entirely novel type of mobile element; iii) the conjugative transposons encoding vancomycin resistance; iv) the integrons, and (v) plasmids which are the principal carrier for mobile and non mobile elements. The GENERAL OBJECTIVES of DRESP are: a) to investigate the molecular epidemiology of the selected families of mobile elements in clinical and environmental situations; b) to carry out structural and functional analysis of these genetic elements to elucidate transfer mechanisms and their regulation, and to identify possible common molecular themes underlying these functions; c) to promote Information-Sharing and dialogue to stakeholders as to increase the social value and impact on pubic health of the knowledge gained.

Problem:

Antibiotic resistance remains a major clinical and public health problem despite the availability of very potent antibiotics. While it is clear that antibiotics are pivotal in the selection of bacterial resistance, the mechanisms of spread of both resistance genes and of resistant bacteria can be very diverse. Mobile DNA provides a major contribution to the spread of antimicrobial resistance, by recruiting new resistance genes in bacterial pathogens and facilitating their horizontal spread among microbial populations. Although the acquisition of new resistance genes is an important factor in the increasing incidence of resistant strains, the nature of the elements carrying the resistance genes and their transfer mechanisms have been clarified only in a few cases, and very little is known about their molecular epidemiology. Moreover, a critical, but often under-appreciated feature of elements carrying resistance genes is their stability: these elements are often able to be integrated into and maintained by new hosts without antibiotic selection, demonstrating that resistance can be very hard to reverse. This feature explains the spread of resistance among community-acquired pathogens not commonly under antibiotic pressure.

A comprehensive understanding of these aspects, beyond the mere epidemiology of resistance genes, could provide a significant breakthrough in combating drug-resistant pathogens by clarifying the dynamics and patterns of spreading of the various mobile elements. This could possibly lead to the identification of new unconventional targets for antimicrobial chemotherapy.

Aim:

DRESP2 is a multidisciplinary project in which the scientific synergism and technical complementarities of the participants is coordinated for a innovative proposal on the characterisation of the molecular mechanism(s) underlying mobility of genetic elements carrying antimicrobial resistance genes. This characterisation will be performed applying up to day post-genomic technologies (Comparative genomics, DNA and RNA micro-arrays, gene chip technology, PCR rep typing, quantitative and high throughput real-time PCR), in addition to more established biomedical (antimicrobial resistance surveillance) and biotechnological approaches (mutant construction and recombinant protein production), to the analysis of selected classes of genetic elements. The mobile genetic elements chosen are the prototype elements responsible for the spread of antimicrobial resistance in the last decade. The genetic elements selected are (i) the double resolvase element responsible for the recent spread of the macrolide efflux determinant (mef(A)) in Gram-positive Streptococci, (ii) the still un-described double resolvase element responsible for beta-lactam resistance in anaerobic Gram-negative cocci, (iii) the integrons responsible for spread of a broad repertoire of resistance genes in Gram-negative bacilli, (iv) the conjugative transposons responsible for spread of vancomycin resistance in Enterococci, and (v) plasmids.

While there are a plethora of resistance determinants, the number of genetic elements responsible for their sensational evolutionary success is limited.
Unfortunately, most industrially financed research focuses on determinants of resistance, mechanisms of resistance, and novel compounds, neglecting the possibly most important feature for public health which is the efficiency of spread which is intimately and exclusively linked to the genetic elements. The DRESP proposal through, (i) fine characterisation of molecular mechanisms behind mobilisation, (ii) cross linking of epidemiological and evolutionary data, and (iii) broad spectrum gene expression profiling, in elements of evolutionary very distant organisms, is expected to produce innovative results. The value of the results of the DRESP proposal, including licensable tools for diagnosis and public health monitoring, will not be restricted to the elements studied, but are expected to add fundamental knowledge to the field of study encompassing all mobile genetic elements and on antimicrobial resistance spread in general.

Expected and obtained results:

During the progress of the DRESP2 project an exceptional amount of data were produced and made available to the scientific community by publication in scientific journals and books and through the project web page. Data range from genomics, to epidemiology, nomenclature and the molecular function of mobile elements. The DRESP2 project produced so far over 60 publications acknowledging specifically this funding source.

Key contributions in this extensive list of data are the description and application of a new techniques for molecular replicon typing of plasmids and the description of antibiotics as signalling agents.

Antibiotics as signals that trigger specific bacterial responses.The ecological and evolutionary roles of antibiotics have been usually inferred from their therapeutical activity. Since those compounds inhibit bacterial growth, it was thought that they should be produced by soil microorganisms to inhibit the growth of competitors in natural habitats. It has been shown however that antibiotics modulate transcription of bacteria in a dose dependent manner. Furthermore, each antibiotic triggers a specific response, and those responses may have adaptive values. From these observations, it has been suggested that antibiotics may have a role as signalling molecules besides inhibitors. The facts that a number of antimicrobials not used for therapy (like lantibiotics) are involved in quorum sensing and that classical quorum sensing autoinducers have antimicrobial activity further support this dual role for antibiotics (Fajardo and Martínez JL Curr Opin Microbiol. 2008. 11(2):161-167).

Replicon typing of plasmids encoding resistance to newer beta-lactams. Polymerase chain reaction-based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems (Carattoli et al., Emerg Infect Dis. 2006. 12(7):1145-8.). This novel technology has bee applied throughout DRESP2 in a variety of collaborations to map in depth the contribution of mobile elements to drug resistance spread and has been published in over fifteen manuscripts.

Potential applications:

in vitro diagnostic medical device
epidemiology of drug resistances
antimicrobial treatment target discovery
evaluation of the impact of treatment schemes
prediction of drug resistance emergence to novel compounds

Coordinators:

Dr. Marco R. Oggioni,Prof Gianni Pozzi, Dr. Gian Maria Rossolini
LA.M.M.B. (Laboratorio di Microbiologia Molecolare e Biotecnologia)
Dipartimento di Bilogia Molecolare
Università degli Studi di Siena
Policlinico Le Scotte (lotto 5, piano 1)
53100 Siena, Italy
Tel.+39 0577233101 (Oggioni); +39 0577233430 (Pozzi); +39 0577233326 (Rossolini)
Fax +39 0577233334
oggioni@unisi.it; pozzi@unisi.it; rossolini@unisi.it

European Commission