Emmanuelle Cambau

Nationwide survey of extended-spectrum β-lactamase-producing Enterobacteriaceae in the French community setting

OBJECTIVES The aim of this study was to assess the prevalence of the extended-spectrum beta-lactamase (ESBL)-producing enterobacteria (ESBLE) in the French community, during a 2006 survey. METHODS All enterobacteria isolated from urine samples of patients, exhibiting a decreased susceptibility to broad-spectrum cephalosporins, were analysed for their beta-lactamase content (synergy test, isoelectrofocusing, conjugation transfer, PCR amplification and/or cloning experiments and sequencing). Additional co-resistances were investigated by PCR, sequencing and/or cloning. Epidemiological relationship was studied by PFGE for all species and, in addition, for Escherichia coli by the determination of the phylogenetic group, multilocus sequence type (ST) and O25b antigen. Characteristics of CTX-M-producing E. coli carriers were compared with other ESBLE carriers. RESULTS Seventy-two ESBLE were collected from 71 patients. Most of them expressed a CTX-M enzyme (n = 42, comprising 40 E. coli), with a predominance of CTX-M-15 (n = 24); 10 CTX-M-15-producing E. coli belonged to the same clone (phylogroup B2, ST131, serotype O25b). The 30 remaining strains possessed a TEM- or SHV-type ESBL. In addition, three strains presented unusual co-resistances such as DHA-1 (n = 2), QnrB4 and ArmA. Risk factors for ESBLE acquisition were substantially less frequent when the ESBL was of the CTX-M type, except for prior antimicrobial therapy. Eighteen percent of the patients were considered to have true community-acquired ESBLE; most of them harboured a CTX-M-producing E. coli. CONCLUSIONS This first nationwide study reports an ESBLE prevalence of 1.1% in the French community setting in 2006, mainly related to the presence of CTX-M-producing E. coli strains; furthermore, unusual co-resistances rarely found in the community setting were occasionally observed, which may threaten future emergence.

Target specificity of the new fluoroquinolone besifloxacin in Streptococcus pneumoniae, Staphylococcus aureus and Escherichia coli

OBJECTIVES Besifloxacin is a new fluoroquinolone in development for ocular use. We investigated its mode of action and resistance in two major ocular pathogens, Streptococcus pneumoniae and Staphylococcus aureus, and in the reference species Escherichia coli. METHODS Primary and secondary targets of besifloxacin were evaluated by: (i) mutant selection experiments; (ii) MIC testing of defined topoisomerase mutants; and (iii) inhibition and cleavable complex assays with purified S. pneumoniae and E. coli DNA gyrase and topoisomerase IV enzymes. RESULTS Enzyme assays showed similar besifloxacin activity against S. pneumoniae gyrase and topoisomerase IV, with IC(50) and CC(25) of 2.5 and 1 microM, respectively. In contrast to ciprofloxacin and moxifloxacin, besifloxacin was equally potent against both S. pneumoniae and E. coli gyrases. DNA gyrase was the primary target in all three species, with substitutions observed at positions 81, 83 and 87 in GyrA and 426 and 466 in GyrB (E. coli numbering). Topoisomerase IV was the secondary target. Notably, resistant mutants were not recovered at 4-fold besifloxacin MICs for S. aureus and S. pneumoniae, and S. aureus topoisomerase mutants were only obtained after serial passage in liquid medium. Besifloxacin MICs were similarly affected by parC or gyrA mutations in S. aureus and S. pneumoniae and remained below 1 mg/L in gyrA-parC double mutants. CONCLUSIONS Although mutant selection experiments indicated that gyrase is a primary target, further biochemical and genetic studies showed that besifloxacin has potent, relatively balanced activity against both essential DNA gyrase and topoisomerase IV targets in S. aureus and S. pneumoniae.

Low selection of topoisomerase mutants from strains of Escherichia coli harbouring plasmid-borne qnr genes

OBJECTIVES To investigate mutations in the type II topoisomerase genes in quinolone-resistant mutants selected from bacteria harbouring plasmid-borne qnr genes. METHODS Mutants were selected by nalidixic acid, ciprofloxacin and moxifloxacin from two Escherichia coli reference strains and corresponding transconjugants harbouring qnrA1, qnrA3, qnrB2 or qnrS1 genes. RESULTS The proportion of resistant mutants selected by the three quinolones was, respectively, in the same range for qnr-positive transconjugants and reference strains. Only 20% (65/329) of the mutants selected from the transconjugants showed a gyrase mutation, whereas 79% (94/119) of those from the reference strains without a qnr gene did (P < 0.0001). At four times the MIC of the selector quinolone, gyrA mutants represented 49% and 95% of the mutants selected with nalidixic acid, 4% and 94% with ciprofloxacin and 0% and 54% with moxifloxacin for qnr-positive transconjugants and reference strains, respectively. Mutations within gyrA were distributed at codon 87 (D87G, H, N or Y) and at codon 83 (S83L) with three novel mutations (gyrA Ser83stop, gyrA Asp82Asn and gyrB insertion of Glu at 465) and three rare mutations (gyrA Gly81Asp, gyrA Asp82Gly and gyrA Ser431Pro), mainly obtained from reference strains after moxifloxacin selection. Strikingly, none of the mutants selected by moxifloxacin from qnr-positive transconjugants harboured a mutation in the topoisomerase genes. CONCLUSIONS Topoisomerase mutants are rarely selected by ciprofloxacin and moxifloxacin from strains harbouring qnr. This suggests that the quinolone resistance-determining region domains are protected from quinolones by the Qnr protein and consequently other mechanisms are developed to acquire a further step of fluoroquinolone resistance.

Sequences of conserved region in the A subunit of DNA gyrase from nine species of the genus Mycobacterium: phylogenetic analysis and implication for intrinsic susceptibility to quinolones.

The sequences of a conserved region in the A subunit of DNA gyrase corresponding to the quinolone resistance-determining region were determined for nine mycobacterial species and were compared. Although the nucleotide sequences were highly conserved, they clearly differentiated one species from another. The results of the phylogenetic analysis based on the sequences of the quinolone resistance-determining regions were compared with those provided by the 16S rRNA sequences. Deduced amino acid sequences were identical within the nine species except for amino acid 83, which was frequently involved in acquired resistance to quinolones in many genera, including mycobacteria. The presence at position 83 of an alanine for seven mycobacterial species (M. tuberculosis, M. bovis BCG, M. leprae, M. avium, M. kansasii, M. chelonae, and M. smegmatis) and of a serine for the two remaining mycobacterial species (M. fortuitum and M. aurum) correlated well with the MICs of ofloxacin for both groups of species, suggesting the role of this residue in intrinsic susceptibility to quinolones in mycobacteria.

Characterization of mutations in Mycobacterium smegmatis involved in resistance to fluoroquinolones.

Fluoroquinolone-resistant mutants of Mycobacterium smegmatis have been obtained in vitro by using ofloxacin as a selecting agent. Two types of mutants were identified according to their quinolone resistance patterns. Type 1 showed a low level of resistance to ofloxacin (MIC of 8 micrograms/ml), whereas a high level of resistance to this drug (MICs of 32 to 64 micrograms/ml) characterized type 2. By using two oligonucleotide primers homologous to DNA sequences flanking the quinolone resistance-determining region (QRDR) in the gyrA gene of Escherichia coli and Staphylococcus aureus, a 150-bp DNA fragment was obtained by PCR amplification from total DNA of two wild-type and five mutant strains of M. smegmatis. The nucleotide sequences of the amplified fragments were determined. The deduced amino acid sequence from the wild-type strains showed ca. 79% similarity with the QRDR in the gyrase A subunit from other gram-positive and gram-negative bacteria. The DNA sequences obtained from the fluoroquinolone-resistant mutants of M. smegmatis exhibited nucleotide modifications compared with the wild-type QRDR. The QRDR from type 1 mutants had a C-T or an A-G transition leading to a change from Ala-83 to Val or Asp-87 to Gly, respectively. The QRDR from type 2 mutants had a Val-83 mutation or both Val-83 and Gly-87 mutations detected in the type 1 mutants. These results suggest that point mutations in the QRDR of the mycobacterial gyrA gene are responsible for acquired quinolone resistance in M. smegmatis. Images

Dihydropteroate Synthase Mutations in the folP1 Gene Predict Dapsone Resistance in Relapsed Cases of Leprosy

Molecular detection was compared with the mouse footpad inoculation test for detection of dapsone resistance in 38 strains of Mycobacterium leprae. Mutations of the folP1 gene (at codons 53 or 55) were found in 6 of 6 strains with high-level resistance, in 3 of 4 strains with intermediate-level resistance, and in 1 of 6 strains with low-level resistance, but not in 22 dapsone-susceptible strains. In cases of infection with strains of M. leprae carrying the folP1 mutation, therapy with dapsone may be replaced by therapy with a fluoroquinolone.

Culture-Independent Prediction of Isoniazid Resistance in Mycobacterium tuberculosis by katG.Gene Analysis Directly From Sputum Samples

BACKGROUND The molecular prediction of isoniazid (INH) resistance in Mycobacterium tuberculosis is hampered by the need for specialized equipment, expertise, high costs, a limited range of detectable mutations, or several of these factors. The rationale for the study was to find a practical alternative and to demonstrate generally valid problems. METHODS AND RESULTS DNA extracted from decontaminated sputum pellets was used to amplify a 0.26 kb target sequence within the katG gene. Mutations of codon 315, frequently found in isoniazid-resistant isolates, could be discriminated in a simple agarose minigel format following an AciI digest of the nested polymerase chain reaction (PCR) product. Within a panel of 22 sputum samples, INH resistance could be predicted in 5 of 10 samples containing isoniazid-resistant M. tuberculosis. The protocol is robust, requires little expertise and no specialized equipment, and provides the test results within 2 days. CONCLUSION The results show the feasibility to rapidly and easily detect mutations highly predictive of isoniazid resistance. Nevertheless, this, like any other molecular resistance prediction test, is affected by often neglected factors, including mutation prevalences, the phenomenon of heteroresistance, and a possible bias toward ones own method.

First detection of plasmid-mediated quinolone resistance in the community setting and in hospitalized patients in Switzerland

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