CRAB - Combating resistance to antibiotics

EC contribution: : € 1.795.500
Duration: : 36 months
Starting date: : 01/01/2006
Funding scheme: : Specific Targeted Research Project
Keywords: : antibiotics, horizontal gene transfer, transposition, conjugation, integron, toxin
Contract/Grant agreement number: : LSHM-CT-2005-019023
Project web-site: : http://www.eucrab.org/

Background:

Combating bacteria with antibiotics is an endless race because bacteria acquire antibiotic resistance (AR) genes easily from unknown environmental sources. We think that an appropriate long-term public health objective would be to elucidate the molecular mechanisms behind the observed AR spread in a concerted strategy targeting the dissemination modules, from AR recruitment to their ultimate acquisition by bacterial pathogens. The focus of our project is to explore a mechanistic approach to combat AR by tackling each of the dissemination modules in this chain - integrons, transposons, conjugative plasmids and stability modules - in a concerted approach. We concentrate on the as yet insufficiently understood aspects of these mechanisms.

Problem:

Antibiotic resistance has represented a serious impediment to antibiotic therapy for as long as antibiotics have been used. Although mutations are responsible for some specific cases of AR, the driving force behind the problem of multiresistance to antimicrobials is gene acquisition by human pathogens. In the past, understanding of AR spread and its control was based largely on a unique approach: the precise description of AR genes presently found in hospitals, and inference from this of the working mechanisms of dispersion. This approach resulted in the accumulation of an impressive knowledge base of individual elements, modules and clones of bacteria that underpin AR dissemination. Nevertheless, and in spite of the vast number of publications on the subject, after over 40 years of study, we are as yet unable to circumvent or even simply restrain AR dissemination.

AR genes are usually carried by transposons, plasmids and other mobile genetic elements, which provide mechanisms assuring their sequestration and dissemination (dispersion modules). Interestingly, in gram-negative species, individual AR genes are often in the form of integron cassettes. Resistance integrons (RI) are frequently found in clinical isolates of resistant Enterobacteriaceae and pseudomonads It is thought that RIs and their cassettes derive from superintegrons (SIs), such as those of Vibrio species. SIs contain several hundred tandem integron cassettes and are carried on bacterial chromosomes.

A coherent picture, which could explain the road to multiple AR, is beginning to emerge. Superintegrons could be considered as the "cradles" of AR since many of the integron cassettes are often silent, and can be subjected to random mutational drift, cassette amplification and, if transferred to the expression region, periodic selection. These proto AR genes therefore contribute to diversity at low cost to the bacterial host. Subsequently, integron cassettes are recruited into integron platforms carried by dispersion modules (transposons, plasmids, genomic islands..) where they can be transferred horizontally to other bacterial species. Insertion sequences (IS), small (<2kb) independently mobile DNA segments capture AR genes or integron platforms in various ways to form transposons. These are subsequently translocated to transmissible genetic elements such as conjugative plasmids. In doing so transposons shuffle genes, modulate their expression and in general contribute to the adaptation of the acquired gene to the pathogenic host. Conjugative plasmids transfer AR genes and establish themselves with variable efficiency in pathogenic bacteria. Finally, plasmids need to be stably inherited once they have established themselves in a recipient cell.

Aim:

The principal aim of CRAB was to explore the mechanisms and process dynamics at work in each of the dissemination modules of the chain of AR genes dissemination- integrons, transposons, conjugative plasmids and stability modules - in a concerted approach. We concentrate on the as yet improperly studied aspects of these mechanisms. Our work will be divided in 4 work package (WP):

WP1: mechanism of integron cassette recombination, signals triggering integrase expression and molecular evolution of gene cassettes.
WP2: Propensities of three major classes of IS to acquire, stabilize and vehicle AR genes and identification of host factors that affect transposition.
WP3: Identification of host factors that affect conjugation, development of in vitro models for conjugation on cultured mammalian cells.
WP4: Identification of novel stabilization systems and their intracellular targets and identification of host factors that trigger these systems

Although the main avenues for AR dissemination can be accounted for by the description above, we still lack both a deep knowledge of many of the detailed mechanisms involved as well as of the influence that different factors (genetic and environmental) have on the modes of AR dissemination and final acquisition by pathogens. In a few cases where a more intimate knowledge of these mechanisms is available, for instance, the mechanisms of plasmid stability, this knowledge is already offering new tools for biotechnology. In summary, we propose to investigate on the mechanisms of AR transmission in the hope of identifying and characterizing targets for novel biotechnological intervention.

Expected and obtained results:

The establishment that the recombination reactions involved in integron cassette insertion and excision rely on an unconventional recombination pathway. This reaction is directed by the presence of two extrahelical bases in the stem-loop structure made from the cassette recombination site under single strand DNA form (partly achieved).
The identification that the general stress response triggers cassette recombination, especially during an antibiotic treatment (achieved).
The determination of the model driving integron cassette evolution (expected).
A description of the different ways by which the three major classes of IS acquire, stabilise and vehicle AR genes (partly achieved).
A quantitative evaluation of the impact of IS elements on horizontal transfer within and between chosen bacterial genera, showing that IS elements moving by replicative transposition are the most efficient (partly achieved).
The precise description of the mechanism of conjugative DNA processing (partly achieved).
The establishment of the host mutations that affect transposition and conjugation (partly achieved).
A list of environmental factors affecting plasmid transfer and biofilm formation on cultured human cells (expected)
The identification of novel stabilisation modules (toxin / antitoxin) and of their functional characterization (partly achieved).
The development of new molecular biological tools (expected)

Potential applications:

Knowledge of cassette recombination process and timing should allow to develop anti-integron molecule or at least improve the antibiotic treatment efficiency.

The application of state-of-the-art functional genomics will ultimately facilitate the translation of genomic data into novel products, such as new stabilization operons, which would be developed by Delphi Genetics.

In addition, several of the novel approaches such as the system of bacterial attachment to biotic surfaces, in situ monitoring of bacterial conjugation and induction of expression of bacterial and plasmid genes, will likely lead to further developments with commercial potential.

Coordinator:

Dr Didier Mazel
Institut Pasteur
Département d structure et dynamique des génomes
Unité Plasticité du Genome Bactérien
25 rue du Dr Roux
75724 Paris, France
Tel. +33 140613284
Fax +33 145688834
mazel@pasteur.fr

European Commission