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  Director : COURVALIN Patrice (pcourval@pasteur.fr)



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 (Bruno Périchon — Marc Galimand)

VanB-type resistance

Acquired resistance to vancomycin in enterococci is most often due to the presence of the vanA and vanB operons. The vanB gene cluster is carried by the Th1549 transposon. Analysis of VanB-type enterococci (14 E. faecium and 4 E. faecalis) isolated from humans in France indicated that (i) all the strains were of the vanB2 subtype, (ii) the vanB2 gene cluster was always associated with Tn1549, and (iii) the transposon was chromosomally located or plasmid borne.

VanE-type resistance

Acquired VanE-type resistance to low levels of vancomycin in E. faecalis BM4405 is due to the inducible synthesis of modified peptidoglycan precursors ending in D-alanine-D-serine. The chromosomally located vanE operon is composed of three resistance genes encoding the VanE ligase, the VanXYE DD-peptidase, and the VanTE serine racemase, and of two regulatory genes coding for a two-component regulatory system, VanRE-VanSE. This operon exhibited 43 to 60% identity with the vanC operon, which is responsible for the intrinsic resistance to vancomycin in E. gallinarum, E. casseliflavus, and E. flavescens. Due to the presence of a stop codon, VanSE was probably not functional. The five genes were shown to be cotranscribed. The three resistance genes are sufficient to confer inducible low-level resistance to vancomycin after cloning in a vancomycin-susceptible E. faecalis strain.

Antibiotic resistance in Streptococcus pyogenes (Marc Galimand)

A parC mutation conferring ciprofloxacin resistance in S. pyogenes.

S. pyogenes is responsible for high rates of morbitidy due to an increase in invasive group A streptococcal infections and bacteremia world-wide during the last decade. In Gram-positive cocci, fluoroquinolone resistance has been associated with mutational alterations in both targets, DNA gyrase and topoisomerase IV, or with active efflux of the drugs. S. pyogenes BM4513 was resistant to low-levels of fluoroquinolones and, as compared to susceptible strains, had a base pair change (TCC/GCC) in the parC gene at position 366 that resulted in amino acid substitution Ser79Ala (S. pyogenes coordinates). The mutation was transformed from the clinical isolate into S. pneumoniae and conferred similar levels of resistance to the new host. These results indicate that massive use of fluoroquinolones can select resistant mutants in non target bacterial species.

A rpoB mutation conferring rifampicin resistance in S. pyogenes.

S. pyogenes BM4478 and Staphylococcus aureus BM4479 were isolated from a patient undergoing rifampicin therapy. High-level resistance to rifampicin was due to the following mutations in the rpoB gene :Ser522Leu in strain BM4478 and His526Asn and Ser574Leu in strain BM4479.

Extended specrum ß-lactamases in Enterobacteriaceae (Thierry Lambert — Marc Galimand)

TEM-103/IRT-28 b-lactamase, a new TEM variant produced by E. coli.

Among the mechanisms responsible for resistance of E. coli to b-lactam-b-lactamase inhibitor combinations, hyperproduction of TEM-1 penicillinase and alteration in the outer membrane proteins limiting the entry of the drugs were first reported. Inhibitor-resistant TEM (IRT) b-lactamases confer resistance to penicillins and their combinations with b-lactamase inhibitors, such as clavulanic acid. IRT variants are derivatives of TEM ß-lactamases with mutations at various positions shown, or postulated, to play a role in determining resistance to inhibitors. Clinical isolate E. coli BM4511 was resistant to various ß-lactams, alone or in combination with b-lactamase inhibitors, but remained susceptible to cephalosporins. Resistance was due to production of a new TEM-type b-lactamase, designated TEM-103/IRT-28, characterized by the Arg275Leu substitution and encoded by the ca. 62-kb pIP845 conjugative plasmid of IncI1 incompatibility group.

ColE1-like plasmid pIP843 of Klebsiella pneumoniae encoding extended-spectrum ß-lactamase CTX-M-17.

Resistance of K. pneumoniae BM4493, isolated in Ho Chi Minh city in Vietnam, to cefotaxime and aztreonam was due to production of the novel CTX-M-17 b -lactamase. The blaCTX-M-17 gene was borne by the 7,086-bp pIP843 plasmid which was entirely sequenced and belonged to the ColE1-family. The 876-bp gene differed from blaCTX-M-14 by two nucleotides that led to the single amino acid substitution Glu289® Lys. blaCTX-M-17 was flanked upstream by an ISEcp1-like element and downstream by an IS903-variant. The transcriptional start site of blaCTX-M-17 was located 109 nucleotides upstream from the initiation codon in the ISEcp1-like element which also provided the promoter sequences. Plasmid pIP843, which was non self-transferable and non mobilisable, contained 5 ORFs. Regions homologous to sequences coding for putative RNAII, RNAI transcripts and a to a rom gene, involved in regulation of transcription and cer-like, responsible for stability of ColE1-like plasmids, were identified. Consensus sequences for putative replication (oriV) and transfer (oriT) origins were present. Results of primer extension experiments indicated that ISEcp1 provides the promoter for expression of blaCTX-M-17 and may contribute to dissemination of this gene.

Distribution of extended-spectrum ß-lactamases in clinical isolates of Enterobacteriaceae in Vietnam.

Among 730 E. coli, 438 K. pneumoniae, and 141 Proteus mirabilis, isolated between September, 2000 and September, 2001 in seven hospitals in Ho Chi Minh city, 27% were resistant to ceftazidime, 30% to cefotaxime, 31% to ceftriaxone, 16% to cefoperazone, and 6% to cefepime. Resistance to imipenem was found in 6% of the isolates. In 55 strains producing extended-spectrum b -lactamases (32 E. coli, 13 K. pneumoniae, and 10 P. mirabilis), structural genes for VEB-1 (25%), CTX-M (25%), SHV (38%), and TEM (76%) enzymes were detected. Sequencing of the PCR products obtained from the K. pneumoniae isolates revealed the presence of blaVEB-1, blaCTX-M-14, blaCTX-M-17, blaSHV-2, and blaTEM-1. Molecular typing of the strains with a similar resistance phenotype to broad-spectrum cephalosporins indicated polyclonal spread. ISEcp1 was presumably responsible for dissemination of the blaCTX-M-like. gene.

Wild-type intracellular bacteria deliver DNA into mammalian cells (Catherine Grillot-Courvalin)

Gene transfer in vitro from intracellular bacteria to mammalian phagocytic and non phagocytic cells and in vivo in mice has been reported. The bacteria used as DNA delivery vectors were engineered to lyse upon entry in the cell due to impaired cell wall synthesis for Shigella flexneri and invasive E. coli or production of a phage lysin for Listeria monocytogenes. In vivo gene transfer was obtained with attenuated Salmonella typhimurium and resulted in stimulation of mucosal immunity. We report that wild type intracellular human pathogens, such as L. monocytogenes EGD or LO28 and S. flexneri M90T, mediate efficient in vitro transfer of functional genes into epithelial and macrophage cell lines. A low-efficiency transfer was obtained from strain EGD to mouse peritoneal macrophages. DNA transfer with S. typhimurium was observed only from attenuated aroA strain SL7207 into COS-1 cell line. As demonstrated by the study of listeriolysin defective L. monocytogenes or of S. typhimurium SL7207 aroA engineered to secrete listeriolysin, escape of bacteria or of plasmid DNA from the intracytoplasmic vacuole is required for transfer of genetic information to occur.

Keywords: bacteriology, antibiotics, resistance, gene transfer

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  Office staff Researchers Scientific trainees Other personnel
  MURGUET Sylvie,smurguet@pasteur.fr

VAN STEENKISTE Pascale,pvanstee@pasteur.fr
GALIMAND Marc, IP, Researcher,galimand@pasteur.fr

GRILLOT-COURVALIN Catherine, M.D., Ph.D., Associate Prof. CNRS,ccourval@pasteur.fr

GUILLEMOT Didier, IP, M.D. Researcher,guillemo@pasteur.fr

MARCHAND Isabelle, IP, Ph.D. Post-Doc,imarchan@pasteur.fr
ABADIA PATIÑO Lorena, Ph.D. student,labadia@pasteur.fr

ALONSO Rodrigo, Ph.D., Post-doctoral fellow

AYOUB Carole, Ph.D. student

BONORA Maria-Grazia, Ph.D. student

CAO Thi Bao Van, Ph.D. student

CATTOIR Vincent, Pharm. resident, DEA

DAHL Kristin, Ph.D., Post-doctoral fellow

GONZÁLEZ-ZORN Bruno, Veterin. D., Ph.D., Post-doctoral fellow,bgzorn@pasteur.fr

KOLBERT Mathias, M.D., Post-doctoral fellow,mkolbert@pasteur.fr

LAMBERT Thierry, Pharm. D., Ph.D., Prof. Univers.,tlambert@pasteur.fr

LEMANISSIER Véronique, Pharm. resident

PANESSO Diana, Ph.D. student

REYNOLDS Peter, Ph.D., Invited Professor

SABTCHEVA Stefana, M.D., Invited Professor

SENNA José Procopio Moreno, Pharm. D., Ph.D. Post-doctoral fellow,senna@pasteur.fr
BERNEDE Claire, Engineer, IP,cbernede@pasteur.fr

CHAUVEL Murielle, Technician, IP,mchauvel@pasteur.fr

DAMIER Laurence, Engineer, IP,ldamier@pasteur.fr

DEPARDIEU Florence, Technician, IP,fdepard@pasteur.fr

GERBAUD Guy, Engineer, IP,

GOUSSARD Sylvie, Technician, IP,sgouss@pasteur.fr

GRONDIN Sophie, Technician, Association Claude Bernard

PERICHON Bruno, Engineer, IP,brunoper@pasteur.fr

SIMON Sylvie, Technician, INSERM

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