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Didier Mazel

Publications and source records attributed to Didier Mazel.

14 recordsLinked to original sources
PubMed2024

Measuring single-cell susceptibility to antibiotics within monoclonal bacterial populations.

The emergence of new resistant bacterial strains is a worldwide challenge. A resistant bacterial population can emerge from a single cell that acquires resistance or persistence. Hence, new ways of tackling the mechanism of antibiotic response, such as single cell studies are required. It is necessary to see what happens at the single cell level, in order to understand what happens at the population level. To date, linking the heterogeneity of single-cell susceptibility to the population-scale response to antibiotics remains challenging due to the trade-offs between the resolution and the field of view. Here we present a platform that measures the ability of individual E. coli cells to form small colonies at different ciprofloxacin concentrations, by using anchored microfluidic drops and an image and data analysis pipelines. The microfluidic results are benchmarked against classical microbiology measurements of antibiotic susceptibility, showing an agreement between the pooled microfluidic chip and replated bulk measurements. Further, the experimental likelihood of a single cell to form a colony is used to provide a probabilistic antibiotic susceptibility curve. In addition to the probabilistic viewpoint, the microfluidic format enables the characterization of morphological features over time for a large number of individual cells. This pipeline can be used to compare the response of different bacterial strains to antibiotics with different action mechanisms.

Anti-Bacterial Agents↗
PubMed2006

Construction of a Vibrio splendidus mutant lacking the metalloprotease gene vsm by use of a novel counterselectable suicide vector.

Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the PBAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.

Animals↗
PubMed2006

Structural basis for broad DNA-specificity in integron recombination.

Lateral DNA transfer--the movement of genetic traits between bacteria--has a profound impact on genomic evolution and speciation. The efficiency with which bacteria incorporate genetic information reflects their capacity to adapt to changing environmental conditions. Integron integrases are proteins that mediate site-specific DNA recombination between a proximal primary site (attI) and a secondary target site (attC) found within mobile gene cassettes encoding resistance or virulence factors. The lack of sequence conservation among attC sites has led to the hypothesis that a sequence-independent structural recognition determinant must exist within attC. Here we report the crystal structure of an integron integrase bound to an attC substrate. The structure shows that DNA target site recognition and high-order synaptic assembly are not dependent on canonical DNA but on the position of two flipped-out bases that interact in cis and in trans with the integrase. These extrahelical bases, one of which is required for recombination in vivo, originate from folding of the bottom strand of attC owing to its imperfect internal dyad symmetry. The mechanism reported here supports a new paradigm for how sequence-degenerate single-stranded genetic material is recognized and exchanged between bacteria.

Attachment Sites, Microbiological↗
PubMed2006

Integrons: agents of bacterial evolution.

Integrons are assembly platforms - DNA elements that acquire open reading frames embedded in exogenous gene cassettes and convert them to functional genes by ensuring their correct expression. They were first identified by virtue of their important role in the spread of antibiotic-resistance genes. More recently, our understanding of their importance in bacterial genome evolution has broadened with the discovery of larger integron structures, termed superintegrons. These DNA elements contain hundreds of accessory genes and constitute a significant fraction of the genomes of many bacterial species. Here, the basic biology of integrons and superintegrons, their evolutionary history and the evidence for the existence of a novel recombination pathway is reviewed.

Attachment Sites, Microbiological↗
PubMed2005

Integron cassette insertion: a recombination process involving a folded single strand substrate.

Integrons play a major role in the dissemination of antibiotic resistance genes among Gram-negative pathogens. Integron gene cassettes form circular intermediates carrying a recombination site, attC, and insert into an integron platform at a second site, attI, in a reaction catalyzed by an integron-specific integrase IntI. The IntI1 integron integrase preferentially binds to the 'bottom strand' of single-stranded attC. We have addressed the insertion mechanism in vivo using a recombination assay exploiting plasmid conjugation to exclusively deliver either the top or bottom strand of different integrase recombination substrates. Recombination of a single-stranded attC site with an attI site was 1000-fold higher for one strand than for the other. Conversely, following conjugative transfer of either attI strand, recombination with attC is highly unfavorable. These results and those obtained using mutations within a putative attC stem-and-loop strongly support a novel integron cassette insertion model in which the single bottom attC strand adopts a folded structure generating a double strand recombination site. Thus, recombination would insert a single strand cassette, which must be subsequently processed.

Attachment Sites, Microbiological↗
PubMed2005

The single-stranded genome of phage CTX is the form used for integration into the genome of Vibrio cholerae.

A major determinant of Vibrio cholerae pathogenicity, the cholera enterotoxin, is encoded in the genome of an integrated phage, CTXvarphi. CTXvarphi integration depends on two host-encoded tyrosine recombinases, XerC and XerD. It occurs at dif1, a 28 bp site on V. cholerae chromosome 1 normally used by XerCD for chromosome dimer resolution. The replicative form of the phage contains two pairs of binding sites for XerC and XerD in inverted orientations. Here we show that in the single-stranded genome of the phage, these sites fold into a hairpin structure, which creates a recombination target for XerCD. In the presence of XerD, XerC can catalyze a single pair of strand exchanges between this target and dif1, resulting in integration of the phage. This integration strategy explains why the rules that normally apply to tyrosine recombinase reactions seemed not to apply to CTXvarphi integration and, in particular, why integration is irreversible.

Base Sequence↗
PubMed2005

A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPalpha) conjugative machineries and their cognate Escherichia coli host strains.

We describe the construction of the pSW family of conditionally replicating plasmids which are based on the IncX oriV origin (oriV(R6Kgamma)) of replication that is dependent on the pir-encoded protein. We constructed several Escherichia coli derivatives expressing pir from different chromosomal loci, and the pir gene could be transduced by phage P1 to any E. coli strain. These chromosomal constructions generate dapA and thyA knockouts, which lead to diaminopimelate or thymidine auxotrophies, respectively, and they serve to provide absolute counterselection even in rich media. These strains can be easily counterselected if used in plasmid transfer experiments into markerless recipients, and they have been demonstrated to work efficiently in E. coli xVibrio or E. coli xBartonella matings. We constructed different pSW plasmids carrying either the oriT(RP4) or the oriT(R388), and we demonstrated that these derivatives can be efficiently transferred using RP4 and R388 conjugation machineries, respectively. We also constructed two plasmids expressing the R388 conjugation machinery, but lacking the oriT(R388). We demonstrated that these plasmids enabled efficient and exclusive transfer of a pSW-oriT(R388) derivative from E. coli to V. cholerae, and we offer an alternative to the popular RP4-based delivery system.

Conjugation, Genetic↗
PubMed2005

Integron-associated antibiotic resistance and phylogenetic grouping of Escherichia coli isolates from healthy subjects free of recent antibiotic exposure.

The study of integrons in 181 Escherichia coli isolates from three groups of healthy subjects who lived in communities and had not taken antibiotics for at least 1 month showed that the presence of integrons was associated with antibiotic resistance and phylogenetic grouping of the bacterial host and dependent on a subject's living environment.

Anti-Bacterial Agents↗
PubMed2005

Comparative study of class 1 integron and Vibrio cholerae superintegron integrase activities.

Superintegrons (SIs) and multiresistant integrons (MRIs) have two main structural differences: (i) the SI platform is sedentary, while the MRI platform is commonly associated with mobile DNA elements and (ii) the recombination sites (attC) of SI gene cassette clusters are highly homogeneous, while those of MRI cassette arrays are highly variable in length and sequence. In order to determine if the latter difference was correlated with a dissimilarity in the recombination activities, we conducted a comparative study of the integron integrases of the class 1 MRI (IntI1) and the Vibrio cholerae SI (VchIntIA). We developed two assays that allowed us to independently measure the frequencies of cassette deletion and integration at the cognate attI sites. We demonstrated that the range of attC sites efficiently recombined by VchIntIA is narrower than the range of attC sites efficiently recombined by IntI1. Introduction of mutations into the V. cholerae repeats (VCRs), the attC sites of the V. cholerae SI cassettes, allowed us to map positions that affected the VchIntIA and IntI1 activities to different extents. Using a cointegration assay, we established that in E. coli, attI1-x-VCR recombination catalyzed by IntI1 was 2,600-fold more efficient than attIVch-x-VCR recombination catalyzed by VchIntIA. We performed the same experiments in V. cholerae and established that the attIVch-x-VCR recombination catalyzed by VchIntIA was 2,000-fold greater than the recombination measured in E. coli. Taken together, our results indicate that in the V. cholerae SI, the substrate recognition and recombination reactions mediated by VchIntIA might differ from the class 1 MRI paradigm.

Base Sequence↗
PubMed2003

Comparative analysis of superintegrons: engineering extensive genetic diversity in the Vibrionaceae.

Integrons are natural tools for bacterial evolution and innovation. Their involvement in the capture and dissemination of antibiotic-resistance genes among Gram-negative bacteria is well documented. Recently, massive ancestral versions, the superintegrons (SIs), were discovered in the genomes of diverse proteobacterial species. SI gene cassettes with an identifiable activity encode proteins related to simple adaptive functions, including resistance, virulence, and metabolic activities, and their recruitment was interpreted as providing the host with an adaptive advantage. Here, we present extensive comparative analysis of SIs identified among the Vibrionaceae. Each was at least 100 kb in size, reaffirming the participation of SIs in the genome plasticity and heterogeneity of these species. Phylogenetic and localization data supported the sedentary nature of the functional integron platform and its coevolution with the host genome. Conversely, comparative analysis of the SI cassettes was indicative of both a wide range of origin for the entrapped genes and of an active cassette assembly process in these bacterial species. The signature attC sites of each species displayed conserved structural characteristics indicating that symmetry rather than sequence was important in the recognition of such a varied collection of target recombination sequences by a single site-specific recombinase. Our discovery of various addiction module cassettes within each of the different SIs indicates a possible role for them in the overall stability of large integron cassette arrays.

Attachment Sites, Microbiological↗
PubMed2002

Bacterial resistance evolution by recruitment of super-integron gene cassettes.

The capture and spread of antibiotic resistance determinants by integrons underlies the rapid evolution of multiple antibiotic resistance among diverse Gram-negative clinical isolates. The association of multiple resistance integrons (MRIs) with mobile DNA elements facilitates their transit across phylogenetic boundaries and augments the potential impact of integrons on bacterial evolution. Recently, ancestral chromosomal versions, the super-integrons (SIs), were found to be genuine components of the genomes of diverse bacterial species. SIs possess evolutionary characteristics and stockpiles of adaptive functions, including cassettes related to antibiotic resistance determinants previously characterized in clinical isolates, which suggest that MRIs and their resistance genes were originally recruited from SIs and their pool of amassed genes. However, the recombination activity of integrons has never been demonstrated in a bacterium other than Escherichia coli. We introduced a naturally occurring MRI (TpR, SulR) on a conjugative plasmid into Vibrio cholerae, a species known to harbour a SI. We show that MRIs can randomly recruit genes directly from the cache of SI cassettes. By applying a selective constraint for the development of antibiotic resistance, we demonstrate bacterial resistance evolution through the recruitment a novel, but phenotypically silent, chloramphenicol acetyltransferase gene from the V. cholerae SI and its precise insertion into the MRI. The resulting resistance profile (CmR, TpR, SulR) could then be disseminated by conjugation to other clinically relevant pathogens at high frequency. These results demonstrate that otherwise phenotypically sensitive strains may still be a genetic source for the evolution of resistance to clinically relevant antibiotics through integron-mediated recombination events.

Amino Acid Sequence↗
PubMed2002

The role of integrons in antibiotic resistance gene capture.

Although recently discovered, integrons have played a primordial role in the evolution of bacterial genomes. They are best known as the genetic agents responsible for the capture and spread of antibiotic resistance determinants among diverse Gram-negative clinical isolates, and this activity is at the root of the antibiotic resistance phenomenon that has evolved over the last 60 years. The discovery of the ancestral chromosomal super-integrons, novel integron classes, and the multitude of gene cassettes they propagate solidify the crucial role of this system in adaptive bacterial evolution. Recent evidence suggests that evolutionarily old genetic recombination mechanisms for gene transfer have been adapted to the new antibiotic environment due to the heavy selective pressure of liberal antibiotic use in human medicine and animal husbandry.

Anti-Bacterial Agents↗
PubMedDate not supplied

A new family of conditional replicating plasmids and their cognate Escherichia coli host strains.

We constructed a family of conditionally replicating plasmids, the pTX1 family, which are based on the IncPalpha oriV origin of replication that is dependent on the trfA-encoded protein. We constructed several Escherichia coli derivatives expressing trfA from different chromosomal loci, which can be transduced by phage P1 to any E. coli strain. The pTX1 plasmids also carry the oriTRP4 origin of transfer, and can be conjugated to E. coli, Vibrio cholerae and likely to a broad range of bacteria from the commonly used donor strains SM10 and S17-1, which sustain replication of the plasmids through the trfA gene carried by their integrated RP4. If TrfA is not provided in trans, these plasmids behave as suicide vectors. As such they can be used as a platform for a variety of applications such as those developed on the popular conditionally replicating plasmids carrying the oriVR6Kgamma origin of replication that is controlled by the Pi protein. Their ability to be used as efficient suicide vectors for gene disruption in V. cholerae has been demonstrated.

Bacterial Proteins↗