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 aminoglycosides in Gram-negative bacilli. It has also developped trans-kingdom gene transfer from bacteria to mammalian cells.

VanB-type glycopeptide resistance in Enterococcus (F. Depardieu in collaboration with A. Kolb, Unité des Régulations transcriptionnelles, Institut Pasteur)

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.

E. faecium clinical isolate BM4524, highly resistant to vancomycin, harbours a chromosomal vanB cluster containing the vanRB and vanSB genes encoding a two-component regulatory system and the vanYBWHBBXB resistance genes that are inducibly co-transcribed from the PRB regulatory and PYB resistance promoters, respectively. We showed previously that the VanSB sensor autophosphorylates and transfers its phosphate to the VanRB regulator. In this study, the transcriptional regulation of the regulatory and resistance genes was analysed and the binding sites of VanRB and phosphorylated VanRB in the PRB regulatory and PYB resistance promoters were compared. Purified VanRB protein was phosphorylated with acetylphosphate (VanRB-P). VanRB-P binds at a single site at position -32.5 upstream from the PRB transcriptional start site and at two sites, at positions -33.5 and -55.5, upstream from that of PYB. VanRB dimerises upon acetylphosphate treatment and thus VanRB-P binds with higher affinity than VanRB to its targets resulting in enhanced transcription. VanRB-P recruits the RNA polymerase and appears more efficient than VanRB in promoting open complex formation at PRB and PYB. The PRB and PYB promoters are coordinately regulated, but in a different fashion. The PRB promoter is able to recruit the RNA polymerase in the absence of VanRB and VanRB-P leading to a low level transcription of the regulatory genes in the absence of induction that allows then to switch on the positive autoregulatory loop for expression of the resistance genes in the presence of vancomycin.

Effect of novobiocin on the expression of VanE-type vancomycin resistance in E. faecalis BM4405 (L. Abadía-Patiño and B. Périchon in collaboration with M. Chippaux, CNRS Marseille)

VanE-type E. faecalis BM4405 is resistant to vancomycin. Derivatives susceptible to this antibiotic were obtained, at a high frequency, after treatment of the strain by novobiocin, an inhibitor of the GyrB subunit of DNA gyrase. Sequencing of the vanE operon of a susceptible derivative, BM4405-1, revealed two mutations leading to substitutions in the VanE D-Ala-D-Ser ligase and the VanRE transcriptionnal regulator. However, cloning of the vanE operon of BM4405-1 into a glycopeptide-susceptible E. faecalis strain conferred resistance to vancomycin, indicating that these mutations were not responsible for susceptibility. Sequencing of the gyrB gene of BM4405-1 revealed a mutation responsible for substitution in GyrB of a residue, K337Y, required for ATPase activity and thus implicated in DNA supercoiling. Cloning of the gyrB gene of BM4405 restored vancomycin resistance to BM4405-1. Alteration of DNA supercoiling could be responsible for lack of expression of the vanE operon and thus for vancomycin susceptibility in BM4405-1.

Characterization of the van glycopeptide resistance operons in Paenibacillus (L. Guardabassi and B. Périchon in collaboration with J. van Heijenoort and D. Blanot University Paris-Sud, Orsay)

The organization and sequence of the van operons in two glycopeptide-resistant Paenibacillus isolated from soil were characterized. Regulatory and resistance genes, homologous to the corresponding genes in enterococcal vanA and vanB operons, were found. In these strains, glycopeptide resistance was inducible by vancomycin and teicoplanin and resulted from the synthesis of peptidoglycan precursors containing diamino-pimelic acid and ending in D-Ala-D-Lac. The close similarity of these operons with those of enterococci support the hypothesis that glycopeptide resistance originated in soil organisms and was subsequently acquired by enterococci.

Clonal spread of pediatric isolates of ciprofloxoxacin-resistant, emm type 6 Streptococcus pyogenes. (M. Galimand in collaboration with R. Alonso, Facultad de Farmacia, Vitoria-Gasteiz, Spain).

Twenty-four community isolates of S. pyogenes resistant to ciprofloxacin and susceptible to other quinolones were studied. Sequence determination of the quinolone-resistance determining regions in the gyrA and parC genes revealed a T/G mutation in parC leading to a Ser79Ala substitution in ParC. All isolates were of the emm type 6 and eighteen and two of them were indistinguishable or closely related, respectively, on the basis of pulsed-field gel electrophoresis. The use of fluoroquinolones can select resistant mutant in non-target bacterial species and are not indicated to treat infections in children. Thus, emergence of clinical isolates resistant to ciprofloxacin, in particular among pediatric patients, is a cause of concern and emphasizes the need for judicious use of antibacterial agents.

Worldwide disseminated armA aminoglycoside resistance methylase gene is borne by composite transposon Tn1548. (M. Galimand, S. Sabtcheva, and T. Lambert)

The armA (aminoglycoside resistance methylase) gene which confers high-level resistance to 4,6-disubstituted deoxystreptamines and to fortimicin by post-transcriptional modification of 16S rRNA was initially found in Klebsiella pneumoniae BM4536 on an IncL/M plasmid pIP1204 of ca. 90 kb which also encodes extended spectrum ß-lactamase CTX-M-3. The armA gene was detected in clinical isolates of Citrobacter freundii, Enterobacter cloacae, Escherichia coli, K. pneumoniae, Salmonella enterica, and Shigella flexneri from various countries where it was always associated with blaCTX-M-3 on an IncL/M plasmid. The armA gene was part of composite transposon Tn1548 together with genes ant3"9, sul1, and dfrXII for resistance to streptomycin-spectinomycin, sulfonamides, and trimethoprim, respectively. The 16.6-kb genetic element was flanked by two copies of IS6 and migrated by replicative transposition. This observation accounts for the presence of armA on self-transferable plasmids of various incompatibility groups and its worldwide dissemination.

In vitro selection of mutants of Streptococcus pneumoniae resistant to macrolides and linezolid : relationship with susceptibility to penicillin G or macrolides. (M. Galimand in collaboration with Archet Hospital, Nice, France)

The rate of acquisition of macrolide resistance by S. pneumoniae did not differ when C-14 and C-16 macrolides were used for selection. Linezolid resistance in pneumococcal strains susceptible to penicillin G and to the macrolides was more difficult to obtain than with macrolides. All linezolid-resistant mutants (30) displayed a mutation in 2 to 4 copies of the 23S rRNA rrl gene, mainly G2576U (27/30) with an additional mutation C2610U observed in certain mutants. Two new mutations, C2612A and C2571G, were also observed. In three linezolid-resistant mutants no mutations were identified within the domain studied, suggesting another mechanism of resistance. Increased resistance to these agents may therefore influence the clinical use of linezolid.

Bacterial transfer of genes into human cells (C. Grillot-Courvalin and S. Goussard, in collaboration with I. Fajac, Hôpital Cochin, France, I. Castagluiolo, Padova, Italy, and D. Schindelhauer, München, Germany)

Expanding their host range, we have shown that the engineered bacterial vectors are able to transfer genes into differenciated pulmonary epithelia, in normal outgrowth of bronchail explant or in cystic fibrosis cell lines. These vectors can also stably propagate and efficiently transfer human artificial minichromosomes ; similarly a genomic construct of 160 kb containing a large portion of the CFTR gene was transfered in HT1080 cells were it was stably transcribed. Finally, in order to use bacteria as vaccination vectors, we have established a mouse model of S. aureus nasal carriage which has allowed study of the host and of bacterial factors involved in colonisation.

National Reference center for Antibiotic Resistance

Evaluation of non-automated techniques for phenotypic detection of VanA-type Staphylococcus aureus (C. Girard-Blanc)

Because of its major importance in public health, we have evaluated the ability of various non automated in vitro susceptibility testing methods to detect glycopeptide resistance in three VanA-type methicillin-resistant S. aureus. To this end, minimal inhibitory concentrations were determined according to the guidelines of the US National Committee for Clinical Laboratory Standards, the Comité de l'Antibiogramme de la Société Française de Microbiologie and the British Society for Antimicrobial Chemotherapy and by using the E-test, agar disc diffusion, agar screening plates, and ATB STAPH galleries. In each of these methods, vancomycin was more efficient than teicoplanin for detecting glycopeptide resistance.

We are responsible for the external quality control of a multicenter study on antibiotic resistance carried out in the international network of the Instituts Pasteur.

We have determined the resistance genotype, in particular to glycopeptides in Gram-positive cocci and to α -lactams in Gram-negative bacilli, of clinical isolates that were sent to the Reference center.

Keywords: bacteriology, antibiotics, resistance, gene transfer


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