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.
1. Glycopeptide resistance in Enterococcus (Florence Depardieu - Bruno Périchon)
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 and VanE) 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.
a) VanD-type resistance. VanD-type strains are constitutively resistant to vancomycin and to low levels of teicoplanin. We studied the organisation of the chromosomal vanD gene cluster in E. faecium 10/96A and the regulation of expression of the resistance genes. The sequence of the vanD gene cluster revealed eight open reading frames. The distal part encoded the VanHD dehydrogenase, the VanD ligase, and the VanXD dipeptidase. Upstream from the structural genes for these proteins was the vanYD gene; a frameshift mutation in this gene resulted in premature termination and accounted for lack of D,D-carboxypeptidase activity. The 5' end of the gene cluster contained the vanRD-vanSD genes coding for a putative two-component regulatory system. A G184S mutation adjacent to the serine involved in the binding of D-Ala1 in the D-Ala:D-Ala ligase (Ddl) led to production of an impaired Ddl and accounts for the lack of D-Ala-D-Ala containing precursors. Vancomycin resistant enterococci having an impaired D-Ala:D-Ala ligase can only grow in the presence of vancomycin if resistance is inducible by vancomycin. In the strain studied, there were no qualitative differences between the peptidoglycan precursors produced by uninduced or induced cells, indicating that the vanD cluster was expressed constitutively, thus bypassing the requirement for glycopeptides. Insertion of ISEfa4, belonging to the IS605-family in the vanSD sensor gene led to constitutive expression of vancomycin resistance.
b) VanG-type resistance. We studied the genetic organisation, the location of the vanG gene cluster, and the regulation of expression of the resistance genes in two VanG-type E. faecalis. The chromosomal vanG cluster was composed of genes recruited from various van operons. The 3' end encoded VanG, a D-Ala:D-Ser ligase, VanXYG, a putative bifunctional D,D-peptidase, and VanTG, a serine racemase as in VanC- and VanE-type strains. Upstream from these structural genes were vanWG with unknown function and vanYG containing a frameshift mutation which resulted in premature termination of the D,D-carboxypeptidase encoded protein. The 5' end of the gene cluster contained three genes vanUG, vanRG, and vanSG encoding a putative regulatory system, which were cotranscribed constitutively from the PUG promoter, whereas transcription of vanYG,WG,G,XYG,TG was inducible and initiated from the PYG promoter. Transfer of VanG-type glycopeptide resistance to another E. faecalis was associated with the movement, from chromosome to chromosome, of genetic elements of ca. 240 kb carrying also ermB which encodes erythromycin resistance. Sequence determination of the flanking regions of the vanG cluster in donors and transconjugants revealed the same 4-bp direct repeats and 22-bp imperfect inverted repeats that delineated the large element.
c) VanB-type resistance. E. faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster including the vanSB/vanRB two-component system regulatory genes. E. faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2-bp insertion leading to an impaired D-Ala:D-Ala ligase. Sequencing of the vanB operon in BM4525 also revealed an 18-bp deletion in the vanSB gene designated vanSB?. The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanSB? histidine kinase leading to constitutive expression of the resistance genes. The VanSB, VanSB? and VanRB proteins were overproduced in E. coli and purified. In vitro autophosphorylation of the VanSB and VanSB? histidine kinases and phosphotransfer to the VanRB response regulator did not differ significantly. However, VanSB? was deficient in VanRB phosphatase activity leading to accumulation of phosphorylated VanRB. Increased glycopeptide resistance in E. faecium BM4525 was therefore due to lack of production of D-Ala-D-Ala ending pentapeptide and to constitutive synthesis of D-Ala-D-Lac terminating peptidoglycan precursors, following loss of D-Ala:D-Ala ligase and of VanSB phosphatase activity, respectively.
d) Influence of VanD Type Resistance on Activities of Glycopeptides In Vitro and in Experimental Endocarditis Due to E. faecium. The consequences of VanD type resistance on the activity of vancomycin and teicoplanin were evaluated in vitro and in a rabbit model of aortic endocarditis with a clinical isolate of E. faecium and its susceptible derivative . The two antibiotics were inactive against the resistant strain in vivo in terms of reduction of bacterial counts and of prevention of emergence of glycopeptide-resistant sub-populations, despite using teicoplanin at concentrations greater than the MIC. This could be due to the high inoculum effect also observed in vitro against both antibiotics. These results suggest that detection of VanD type resistance is of major importance because it abolishes in vivo glycopeptide activity and allows the emergence of highly resistant mutants.
e) Multiple antibiotic resistance gene transfer from animal to human enterococci in the digestive tract of gnotobiotic mice. Enterococci that harbor resistance genes are common in the digestive tract of animals. It has been suggested that these bacteria might serve as a reservoir of resistance genes for human digestive microflora. We have shown that various resistance genes, in particular to the glycopeptides, can be conjugatively transferred from an E. faecium of animal origin to a human strain of the same species in the gastrointestinal tracts of gnobiotic mice in the absence of selective pressure. The ease with which we were able to obtain gene transfer from animal to human enterococci suggests that gene exchange under natural conditions might take place more commonly than suspected.
Fluoroquinolone resistance in Listeria monocytogenes by efflux (Marc Galimand). L. monocytogenes is widely distributed in the environment and can cause serious human infections, primarily in neonates and immunocompromised adults. Food-borne transmission is recognized as the main route of acquisition of the disease which differs from most food-borne infections by its high fatality rate (20%-30% of cases). Although fluoroquinolones do not include listeriosis in their indications they can, due to their increasing use, select resistant Listeria. In Gram-positive bacteria, resistance results from mutational alterations in the intracellular targets of fluoroquinolones, the type II DNA topoisomerases IV or from active export of the drugs via efflux pumps. Five L. monocytogenes isolated from human cases of listeriosis in France were found to be resistant to fluoroquinolones during screening of 488 L. monocytogenes. Based on a ? 4-fold ciprofloxacin resistance decrease in the presence of reserpine, resistance was attributed to active efflux of the drugs. Comparative analysis of the sequences deduced from the genome of L. monocytogenes EGD with those of the proteins of the Major Facilitator Superfamily allowed us to identify the lde gene which encodes a putative efflux pump. Insertional inactivation of the lde gene indicated that the corresponding protein was responsible for fluoroquinolone resistance. These data indicate that massive use of fluoroquinolones can select resistant mutants in non target bacterial species.
2. Resistance to aminosides in Gram-negative bacilli (Marc Galimand)
Despite the development of new ß-lactams and fluoroquinolones, aminoglycosides are still used for the treatment of severe Gram-negative infections. There are three known mechanisms of resistance to aminoglycosides in human pathogens: (i) decreased intracellular accumulation of the antibiotic by alteration of the outer membrane permeability, diminished inner membrane transport, or active efflux, (ii) modification of the target by mutation in ribosomal proteins or in 16S RNA, and (iii) enzymatic modification of the drug which is the most common. Micro-organisms that produce aminoglycosides have developed an additional pathway to avoid suicide which involves post-transcriptional methylation of ribosomal RNA.
a) Plasmid-mediated high-level resistance in Enterobacteriaceae by 16S rRNA methylation.
Aminoglycosides bind to a highly conserved motif of 16S RNA which leads to alterations in the ribosome functions. Self-transferable plasmid pIP1204 conferred multiple antibiotic resistance to a clinical isolate of Klebsiella pneumoniae and resistance to nearly all aminoglycosides was accounted for by a new gene designated armA (aminoglycoside resistance methylase). The cloning of armA in E. coli conferred to the new host high level resistance to 4-6-disubstituted deoxystreptamines. The deduced sequence of ArmA displayed from 37 to 47% similarity to those of 16S rRNA m7G methyltransferases from various actinomycetes which confer resistance to aminoglycoside producing strains. It therefore appears that post-transcriptional modification of 16S rRNA can confer high-level broad-range resistance to aminoglycosides in Gram-negative human pathogens. The armA gene was flanked by insertion sequences and linked to blaCTX-M which confers resistance to cephalosporins by synthesis of an extended spectrum ß-lactamase. These elements favor dissemination of armA and it is thus not surprising that we found the gene in various Enterobacteriaceae isolated from several european countries
b) Characterization of the ant(4')-IIb gene in Pseudomonas aeruginosa.
Aminoglycosides are antibiotics of major importance in the treatment of infections due to P. aeruginosa. Amikacin resistance is due to production of either 6'-N-acetyltransferase I, 3'-O-phosphotransferase VI, or 4'-O-nucleotidyltransferase type II [ANT(4')-II]. The ANT(4')-II enzyme in P. aeruginosa confers resistance to amikacin, and to other aminoglycosides with a 4'-hydroxyl group. We have studied P. aeruginosa strains isolated in Bulgaria which were resistant to amikacin and did not harbor the ant(4')-IIa gene. The ant(4')-IIb gene was identified as a coding sequence of 756 bp. Alignment of ANT(4')-IIb with ANT(4')-IIa indicated that the first 156 amino acids were conserved. The similarity was interrupted at Arg205 whereas homology at the nucleotide level was conserved throughout the genes. This was due to an additional guanosine at position 1677 of the deposited sequence leading to a frame shift mutation. This result suggests that the C terminal portion of ANT(4') enzymes may not be required for activity. In order to test this hypothesis, a PCR fragment encoding a protein corresponding to ANT(4')-IIa but without the 43 C-terminal amino acids was cloned in an expression vector and found to confer aminoglycoside resistance to E. coli. These data suggest that the carboxy-terminus was required for enzyme activity but that the amino acid sequence could be altered.
Keywords: bacteriology, antibiotics, resistance, gene transfer