Unit: Antibacterial Agents

Director: COURVALIN Patrice

The Antibacterial Agents Unit studies the genetic support, biochemical mechanisms, heterospecific expression , evolution and dissemination of antibiotic resistance in bacterial pathogens for humans ; in particular : enterococci and glycopeptides, and resistance to ß-lactams and aminoglycosides in Gram-negative bacilli. It has also developped trans-kingdom gene transfer from bacteria to mammalian cells.

Glycopeptide resistance in Enterococcus (Florence DEPARDIEU and Bruno PERICHON)

Glycopeptide resistance in enterococci results from the production of modified peptidoglycan precursors ending in D-alanyl-D-lactate (D-Ala-D-Lac) (VanA, VanB, and VanD) or D-Ala-D-serine (VanC, VanE, and VanG) to which glycopeptides exhibit low binding affinities and from the elimination of the high affinity D-Ala-D-Ala-ending precursors synthesized by the host Ddl ligase.

VanD-type resistance

VanD-type strains are constitutively resistant to vancomycin and to low levels of teicoplanin by synthesis of peptidoglycan precursors terminating in D-Ala-D-Lac. We have studied the organisation of the chromosomal vanD gene cluster in recently isolated strains of Enterococcus faecium and for the first time of E. faecalis and the regulation of expression of the resistance genes. Each clinical isolate had lost the chromosomal D-Ala:D-Ala ligase activity following various mutations (E13G implicated in D-Ala 1 binding, S319N at the serine implicated in the ATP binding, or a 7-bp insertion at various locations). Vancomycin resistant enterococci having an impaired D-Ala:D-Ala ligase can only grow in the presence of vancomycin since they rely on the inducible resistance pathway for peptidoglycan synthesis. In the strains studied, there were no qualitative differences between the peptidoglycan precursors produced by uninduced or induced cells, indicating that the vanD cluster was expressed constitutively. Various mutations in the VanSD sensor (insertion of 7 bp or deletion of 1 bp leading to a putative truncated protein) or, for the first time, in the VanRD transcriptional activator (E140G substitution in the effector domain) led to constitutive resistance to vancomycin.

VanB-type resistance

VanB-type strains are inducibly resistant to vancomycin but susceptible to teicoplanin, since the latter drug is not an inducer. Mutations in the vanSB gene allow for constitutive or teicoplanin-inducible expression of the resistance genes. To analyze expression of the van genes in rabbits with experimental endocarditis, a VanB-type E. faecalis with a transcriptional fusion between the PYB promoter for the resistance genes and the gfp mut1 gene for the green-fluorescent protein was constructed in the chromosome of the host. The reporter system allowed monitoring of the expression of the glycopeptide resistance genes in vivo at a single cell level.

VanE-type resistance

VanE-type strains are characterized by low-level resistance to vancomycin and susceptibilty to teicoplanin. We have studied three additional strains of E. faecalis harbouring the vanE operon isolated in Australia. Expression of the resistance genes was inducible in two strains and constitutive in the remaining isolate, probably because of a 2-bp deletion into the vanSE gene leading to a frameshift and truncated protein.

VanA-type glycopeptide resistance in Staphylococcus aureus

Two methicillin- and vancomycin-resistant S. aureus strains (MI-VRSA and PA-VRSA) have been recently isolated. Whereas MI-VRSA is highly resistant to vancomycin and teicoplanin PA-VRSA displays low-level resistance to glycopeptides . We have shown full expression of the vanA operon in the two strains. Resistance is stable in MI-VRSA and highly unstable in PA-VRSA. Induction of resistance by vancomycin was significantly delayed in PA-VRSA relative to MI-VRSA. Low-level glycopeptide resistance of S. aureus PA-VRSA is thus likely due to instability of the genetic element, plasmid or transposon, carrying the vanA operon associated with a longer lag phase before growth resumes after induction by vancomycin.

Multiplex PCR for detection of glycopeptide resistance

As just mentioned, VanA-type resistance to glycopeptides has disseminated to methicillin-resistant S. aureus and certain strains escape phenotypic detection by automated rapid methods. We therefore developed a multiplex PCR assay for detection of the six types of glycopeptide resistance characterized in enterococci and for identification of the most important enterococci and staphylococci at the species level. Primers targeting genes vanA, vanB, vanC, vanD, vanE, and vanG, ddl of E. faecium and E. faecalis, nuc of S. aureus, and a chromosomal portion specific to S. epidermidis were designed to allow amplification of fragments with various sizes in a single reaction.

A PCR-RFLP Assay for Detection of Mutations in Fluoroquinolone-resistant Streptococcus pneumoniae. (R. Alonso and M. Galimand)

Quinolone resistance in S. pneumoniae is mainly due to mutations in the quinolone resistance-determining regions (QRDR) of either the ParC subunit of topoisomerase IV or the GyrA subunit of DNA gyrase. We have developed a PCR-RFLP assay to detect mutations in the Ser79 and Asp83 codons of parC, Asp435 of parE, and Ser81 and Glu85 of gyrA genes which lead to quinolone resistance.

S. pneumoniae CP1000 and 25 in vitro and in vivo resistant derivatives, with known mutations in the QRDR of the parC, parE, and gyrA genes, were used to validate the assay. Mutations within parC were detected using restriction endonucleases HinfI [Ser79] and LweI [Asp83], that in parE [Asp435] with HinfI, and those within gyrA by HinfI [Ser81] and MboII [Glu85], an artificially generated recognition site. The 366-bp parC product from CP1000 contained two HinfI and one LweI cutting sites generating fragments of 183, 127, and 56 bp and of 224 and 142 bp, respectively. Loss of a HinfI site following mutations at the Ser79 codon generated two 183-bp fragments running as a doublet. Mutations at the Asp83 codon suppressed the LweI site and a 366-bp fragment was observed. The 290-bp parE product from CP1000 contained two HinfI sites generating 166-, 87-, and 37-bp fragments. Loss of one of the HinfI sites following mutations at the Asp435 codon generated fragments of 203 and 87 bp. The 183-bp gyrA product from CP1000 contained a natural HinfI site and an artificially created MboII site generating fragments of 113, and 70 bp and of 141 and 42 bp, respectively. Loss of the HinfI site in resistant isolates due to mutations at the Ser81 codon led to a 183-bp fragment. Mutations at the Glu85 codon were associated with loss of the MboII site leading also to a 183-bp fragment.

The rapid and simple assay developed could be a useful screening tool for the most common mutations and facilitate epidemiological studies on resistance to quinolones in clinical isolates of S. pneumoniae.

Resistance to antibiotics by efflux in Acinetobacter baumannii (Laurence Damier-Piolle and Thierry Lambert)

A. baumannii clinical isolate BM4454 was resistant to all aminoglycosides and did not produce an antibiotic inactivating enzyme. The adeABC genes have been identified and implicated in this resistance phenotype, as well as in the basal level of resistance to other drugs, including fluoroquinolones, tetracyclines, chloramphenicol, erythromycin, and trimethoprim. The deduced product of the adeB gene was homologous to membrane proteins of the " Resistance Nodulation cell Division " (RND) family. The adeA and adeC genes encoded a fusion protein and an outer membrane protein, respectively. The AdeA, AdeB, and AdeC proteins associate in a tripartite complex to form a RND-type efflux pump, that allows export of the various substrates outside of the cell. Upstream from the adeABC operon, the adeRS genes encoded proteins similar to regulators and sensors of two component systems, respectively. Inactivation of adeS led to aminoglycoside susceptibility. Gentamicin-resistant mutants constitutively expressing the AdeABC pump were obtained in vitro from susceptible A. baumannii strain CIP70-10. These mutants had either a Thr153->Met mutation in AdeS, or a Pro116->Leu mutation in AdeR. These data indicate that expression of the AdeABC pump is under the control of the AdeRS two component system. Another efflux pump AdeIJK has been identified in A. baumannii BM4454 and its characterization is under process.

Transfer of genes and proteins to mammalian cells by a bacterial vector (C. Grillot-Courvalin and S. Goussard)

We have shown that bacterial vector E. coli dap- BM2710 harboring plasmid pGB2 inv-hly, which confers to the host the ability to invade non-phagocytic cells provided they express ß1-integrins, could transfer functional genes to airway epithelial cell lines or to primary bronchial cells. Confocal and electronic microscopy of the intracellular trafficking of the bacterial vector showed that phagosome-containing E. coli rapidly mature into phagolysosomes. Protein delivery could also be achieved in vitro in cell lines and in vivo in mouse tumor models ; functional E. coli proteins provided a therapeutic benefit when associated with intra peritoneal delivery of 6-MPDR prodrug. We have also demonstrate that under certain conditions, eukaryotic promotors can express bacterial genes.

Keywords: bacteriology, antibiotics, resistance, gene transfer

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