New glycopeptide resistance determinant in enterococci.
E. faecalis BM4405 strain, resistant to vancomycin and susceptible to teicoplanin, synthesizes peptidoglycan precursors ending by D-Alanine-D-Serine. vanE operon, responsible for this resistance, has been assigneed to a chromosomal location and caracterised. It is constituted by three genes which are necessary to the resistance vanE, vanXYE, and vanTE,, coding for a ligase, a D,D-peptidase, and a serine racemase, respectively, and two genes vanRE/vanSE encoding a two-component regulatory system. The organisation of the vanE operon is similar to that of vanC, responsible for the intrinsic resistance to glycopeptides in E. gallinarum and E . casseliflavus-flavescens. This raises the question of the mechanism for interspecific horizontal gene transfer of an entire " house keeping " operon among enterococci. This will be the first addressed by determination of the sequence of the region flanking the vanE gene cluster.
Characterization of transposon Tn1549 conferring VanB-type resistance in Enterococcus spp.
Transfer of VanB-type resistance to glycopeptides, resistance to vancomycin not associated with resistance to teicoplanin, among enterococci has been reported to be associated with the movement of large chromosomal genetic elements or of plasmids. Determination of the flanking regions of the vanB operon allowed the characterization of the 34 kb transposon Tn1549 borne by a plasmid related to pAD1 in clinical isolates of Enterococcus spp. Tn1549 contained thirty open readig frames (ORF) and appeared to be organized like the Tn916 family of conjugative transposons into three functional regions: (i) the right end implicated in the excision-integration process; (ii) the central part, in which the vanB2 operon replaces the tet(M) gene; and (iii) the left extremity, in which eight of the eighteen ORF could be implicated in the conjugative transfer. This type of genetic element could account for the rapid dissemination of vancomycin resistance in enterococci.
Identification of enterococci at the species level by sequencing of the genes for D-alanine:D-alanine ligases.
A PCR assay based on the use of degenerate oligodeoxyribonucleotides allowed characterization of a fragment internal to the ddl genes encoding D-alanine:D-alanine ligases in Enterococcus columbae, E. durans, E. malodoratus, E. mundtii, E. raffinosus, E. seriolicida, E. solitarius, and E. sulfureus. Phylogenetic analysis of the sequence of the amplification products and of those already obtained from E. avium, E. casseliflavus, E. cecorum, E. dispar, E. faecalis, E. faecium, E. flavescens, E. gallinarum, E. hirae, E. pseudoavium, and E. saccharolyticus yielded an evolutionary tree with a topology similar to that based on 16S rRNA sequences. Partial sequencing of the ddl gene can therefore be used for genotypic identification of Enterococcus spp.
Emergence of carbopenem resistance in Klebsiella pneumoniae.
K. pneumoniae clinical isolate BM2974, from Sweden, was resistant to antibiotics including imipenem. Imipenem resistance results from the combination of two resistance mechanisms plasmid-mediated production of CMY-4 cephalosporinase and loss of a major outer membrane protein of ca. 40 kDa.
Molecular characterization of integrons in Acinetobacter baumannii.
Acinetobacter is a bacterial genus responsible for nosocomial infections and multiresistant to antibiotics. Analysis of integrons in A. baumannii clinical isolates indicated a high prevalence of class 1 integrons whereas a class 2 integron was detected in a single strain. The aac(3)-Ia, ant(2")-Ia, aac(6')-Ib, and oxa20 cassettes and composite structures containing insertion sequences were detected. In addition, a hybrid integron composed of a class 2 integrase and a 3' conserved segment of class 1 integrons was characterized. These results indicate that integrons play a major role in multidrug resistance in A. baumannii.
Gene transfer from bacteria to mammalian cells
Certain species of bacteria have evolved the ability to enter mammalian cells by inducing their own internalization. We have shown that invasive strains of Escherichia coli that undergo lysis upon entry into mammalian cells because of impaire a cell wall synthesis can act as stable DNA delivery systems to heir host. This direct transfer of genetic information is efficient, of broad host cell range and the replicative or integrative vectors so delivered are stably inherited and expressed by the cell progeny. Transfer is also observed, although at lower efficiency, from bacterial vectors to digestive or pulmonary differenciated epithelia. DNA delivery to abortive invasion of eukaryotic cells by bacteria is of potential interest for stimulation of mucosal immunity and for in vivo or ex vivo gene therapy of human diseases.
Bactericidal activity of gentamicin against Enterococcus faecalis in vitro and in vivo.
The activity of gentamicin at various concentrations against two strains of Enterococcus faecalis was investigated in vitro and in a rabbit model of aortic endocarditis. In vitro, gentamicin at 0.5 to 4 times the minimal inhibitory concentration (MIC) failed to reduce the number of bacteria at 24 h. Rabbit or human serum dramatically increased gentamicin activity, when the drug concentration exceeded the MIC. Susceptibility testing in the presence of serum was predictive of in vivo activity; however, gentamicin selected resistant mutants in rabbits. The intrinsic activity of gentamicin should be taken into account in the evaluation of combinations of gentamicin and cell wall-active agents against enterococci.
In vitro activity of quinupristin/dalfopristin against worldwide clinical isolates of staphylococci
We have evaluated the in vitro activity of quinupristin/dalfopristin, a streptogramin combination, in comparison with five antibiotics against worlwide clinical isolates of staphylococci. A multicenter in vitro study was performed on fresh, clinically significant, non repetitive strains of staphylococci from patients hospitalized in 23 hospitals in 18 countries. The streptogramin combination showed an excellent in vitro activity against all staphylococcal species tested regardless of the resistance pattern to other drug classes, particularly resistance to methicillin. The synergy of the combination was conserved against macrolide-resistant strains.
National Reference Center for Antibiotics
The major roles of the National Reference Center for Antibiotics on the study of the prevalence of antibiotic resistance in bacterial genera pathogenic for humans ; the maintenance of collections for reference strains, nucleic acid probes, and resistance genes ; the evaluation of the in vitro activity of new antibiotics ; the evaluation and improvement of techniques or automated systems for detection of antibiotic resistance or resistance mechanisms ; and the development of reference techniques for detection of resistance genes.