Sequencing and comparative genomics of Escherichia coli plasmids: a way to understand the evolutionary success of extended-spectrum ß-lactamases – RepliColScope

The spread of antibiotic-resistant pathogens is becoming an extremely serious clinical and public health problem worldwide. In recent year, we have observed a dramatic increase of the resistance to the third generation cephalosporins that are regularly used for empirical therapy of Enterobacteriaceae. Acquired resistance to third generation cephalosporins is mainly mediated by extended-spectrum beta-lactamases (ESBLs) that confer resistance to all beta-lactams except carbapenems and cephamycins. Currently, the high prevalence of all ESBL coding genes, mainly carried on plasmids, is caused by different mechanisms, not mutually exclusive: (i) horizontal transfer of ESBL genes between related or unrelated plasmids, (ii) horizontal transfer of plasmids among unrelated clones and (iii) spread of local or international epidemic clones. During the 1990s, TEM-ESBLs and SHV-ESBLs were dominant. Since the 2000s, CTX-M enzymes have become the most prevalent ESBLs worldwide. Escherichia coli, a versatile human and animal major opportunist pathogen which was up to now sensitive to third generation cephalosporins, has been recognise as the major source of ESBLs. ESBL-producing E. coli are commonly isolated in human community or hospital infection and from faecal carriage and are also increasingly detected in food-producing animals, companion and wild animals and in the environment.
The sudden emergence of E. coli producing ESBLs and the prominence of CTX-M enzymes cannot be explained simply as a result of selective pressure exerted by the use of third generation cephalosporins. Other factors may influence this evolution at ecological and/or molecular levels.
The objective of this project is to understand the evolutionary success of the ESBLs in
E. coli, and especially the CTX-M enzymes emergence as compared to the TEM and SHV enzymes.
To reach this objective, we propose (i) to sequence 80 E. coli plasmids, carrying the three major types of ESBLs (70 plasmids from human isolates and 10 plasmids from animal isolates) and 20 comparative plasmids from periods before the use of third generation cephalosporins (17 plasmids from the ECOR collection strains and 3 plasmids from the E. coli strains of the Murray collection dating from the pre-antibiotic period), (ii) to perform comparative genomics with the generated and already available data and (iii) to test in vitro some hypotheses generated from the in silico data.
The plasmids will be sequenced using a strategy involving high-density pyrosequencing (A 454 Titanium) allowing reads of 400 bp. Novel strategies will be developed in term of automatic structural and functional annotation of the plasmid sequences and the results will be integrated into a special database to perform relevant expert annotations and to explore the data. Comparative genomic and phylogenetic analyses will be performed to reconstruct the evolutionary history of the plasmid genes by identifying those having the most similar gene content. The data will also be analysed in the light of the chromosomal genetic background, represented by the phylogenetic history of the strains and the CRISPRs. We will explore the possible cross talk between modules carried on plasmids and those found on the chromosomes of E. coli, which can play a key role in the stabilization of these extrachromosomal elements.
This project should provide us amounts of quantitative and qualitative data that will bring both fundamental and medical insights on plasmid evolution and on antibiotic resistance. Our project could also offer opportunity for advances in new strategies to prevent resistant plasmid dissemination in bacteria.
ReplicolScope is an association between teams that have a strong expertise on the evolutionary biology of E. coli (U722), the epidemiology of the ESBLs among human and animal strains (UR8 and UR1282), the bacterial plasticity (UPGB) and, a team that is skilled in gene sequencing and in bioinformatics (Genoscope).