gyrA mutations in high-level fluoroquinolone-resistant clinical isolates of Escherichia coli.

Double mutations in the quinolone resistance determining region of the gyrase A gene (gyrA) have recently been reported to be associated with high-level resistance to fluoroquinolones in clinical isolates of Escherichia coli. We examined the type and frequency of such mutations in a large number of clinical isolates that were obtained from ten different geographical locations and had been genotypically characterized by pulsed field gel electrophoresis (PFGE) of chromosomal DNA digests. Of 36 isolates with ofloxacin MICs > or = 4 mg/L that represented at least 24 distinct genotypes, 35 had double mutations at amino acid codons 83 and 87 of gyrA, while two isolates with ofloxacin MICs of 0.5 and 4 mg/L, respectively, each had a single mutation at codon 83. Mutations at codon Ser-83 were uniform, resulting in substitution by Leu. The additional mutations at amino acid codon 87 in the 35 double-mutants were diverse, resulting in Asp-87 substitutions by residues Asn (23 isolates), Gly (7 isolates), Tyr (4 isolates), or His (1 isolate) without a discernable correlation with fluoroquinolone MICs or with phenotypic resistance to chemically unrelated antibacterial agents. Maximal differences between MICs of double-mutants with the same amino acid substitution were eight-fold. The changes of amino acid residues at codon Asp-87 differed between individual patient isolates with the same genotype (and similar MICs), suggesting that the amino acid codon 87 mutations (and possibly the development of high-level fluoroquinolone resistance) might have occurred after the transmission and sharing of a precursor strain carrying the Ser-83-->Leu mutation.

Molecular epidemiology of fluoroquinolone-resistant Escherichia coli bloodstream isolates from patients admitted to European cancer centers.

Previous reports have suggested an increasing incidence of highly fluoroquinolone-resistant Escherichia coli causing bacteremia among cancer patients on prophylactic therapy. We used genotyping by pulsed-field gel electrophoresis of chromosomal DNA digests and random amplified polymorphic DNA fingerprinting to study clonal relationships among such isolates obtained at 10 cancer centers located across Europe and the Middle East. Analysis by both methods indicated that isolates from different centers were genotypically unrelated to each other. There were five centers from which more than one individual patient isolate was available, and most demonstrated significant within-center genetic diversity of strains. Strains shared among patients could be identified at two centers. At the center with the largest number of bloodstream isolates from cancer patients available, fluoroquinolone-resistant control isolates from surgical patients and fluoroquinolone-susceptible control isolates from patients admitted to medical services during the same time period were unrelated to resistant cancer patient isolates and to each other as well. A substantial number of fluoroquinolone-resistant isolates (19 of 58) were nontypeable by pulsed-field gel electrophoresis. Fluoroquinolone resistance was commonly associated with multiple antibiotic resistance to chemically unrelated antibacterial agents irrespective of the origin of the isolates.

Characterization of coagulase-negative staphylococci causing nosocomial infections in preterm infants.

The species spectrum, antibiotic susceptibility, and genomic profile of coagulase-negative staphylococci (CNS) isolated from infected preterm infants were compared with those obtained in CNS from nursery personnel. Staphylococcus epidermidis was the predominant species in the 66 investigated preterm infants (171 isolates), accounting for 64% of all isolates. A high proportion of Staphylococcus haemolyticus (32%) could be detected. In contrast to the results in patients, the spectrum in nursery personnel was broad and included more species of CNS. All isolates of CNS from preterm infants demonstrated a low rate of susceptibility to the beta-lactam antibiotics (2% sensitivity to penicillin and 6% sensitivity to oxacillin). Sensitivity to gentamicin (9%) was also rare. An unexpected observation was susceptibility to teicoplanin in only 70% of all CNS isolated from patients due to the high proportion of Staphylococcus haemolyticus. Analysis of the genomic profile of 33 isolates of Staphylococcus haemolyticus by pulsed-field gel electrophoresis revealed a relationship between the strains. An outbreak of one particular strain of Staphylococcus haemolyticus in the neonatal intensive care unit investigated can therefore not be excluded.

Terminal redundancy and circular permutation of mycoplasma virus L3 DNA.

This communication reports the physical map of mycoplasma virus L3 (MV-L3) DNA derived from restriction patterns obtained by digestion with seven different restriction endonucleases. The length of the restriction map is 36,200 bp in contrast to the contour length of native MV-L3 DNA molecules which is 39,400 bp as determined by electron microscopy. The difference in length of 3,200 bp (corresponding to 8.1% of the native viral DNA contour length) is explained by terminal redundancy. It was possible to clone all fragments from particular restriction patterns into Escherichia coli vector pAT153, an indication of circular permutation within a population of MV-L3 DNA. However clear evidence has been obtained from the molar ratios of fragments and from hybridization experiments. We suppose that viral DNA is packaged from a concatemeric precursor molecule starting at a specific site called pac.

Release of mycoplasmavirus L1 upon transfection of Acholeplasma laidlawii with homologous and heterologous viral DNA.

This communication reports on the release of Mycoplasmavirus L1 after infection of Acholeplasma laidlawii with purified L3 virus. Release also occurred after transfection with certain restriction fragments from MV-L3 and MV-L1 genomes. Since circular molecules are efficiently taken up in polyethylene glycol-mediated transfection, inducing fragments were applied cloned in E. coli plasmids. Release was also observed after electroporation of cells incubated with MV-L1 replicative intermediate DNA and linear MV-L3 DNA isolated from virus particles, respectively. Released MV-L1 viruses were identified after virus plaque formation on indicator lawns according to plaque morphology and hybridization with labeled viral DNA probes as well as by DNA restriction analysis. Uninfected and untransfected cells from six laboratory strains of A. laidlawii (including a MV-L1 resistant one) were examined for the presence of MV-L1 DNA. They all bear MV-L1 DNA integrated in their genomes.

[Cervicofacial actinomycosis. Apropos of 2 atypical locations]. / Actinomycose cervico-faciale. A propos de deux localisations atypiques.

Cervico-facial actinomycosis is a chronic bacterial disease caused by actinomycetes. The diagnosis may be made on the specific anatomo-pathological appearance of the granulomas and be confirmed by anaerobic culture of the germs. The authors report two atypical cases: the first is a case of a thyroglossal pseudocyst, the second that of adenoidal vegetations in an adult. Treatment with Penicillin G or V for six weeks produced a favourable response. The various clinical forms of the condition described in the literature, should encourage the physician to include bacteriological testing for actinomycetes among his investigations.

Electroporation-mediated transfection of Acholeplasma laidlawii with mycoplasma virus L1 and L3 DNA.

In contrast to mycoplasma virus L1 and L2 circular DNA, mycoplasma virus L3 linear DNA is not biologically active in polyethylene glycol-mediated transfection. Electroporation of Acholeplasma laidlawii, however, leads to plaque formation after incubation with L3 DNA. The efficiency of electroporation-mediated transfection is 1/10 that of polyethylene glycol-mediated transfection as estimated with L1 DNA. Trypsin treatment of cells before DNA addition increases the efficiency of DNA uptake.

Increased amounts of small polydisperse circular DNA (spcDNA) in angiofibroma-derived cell cultures from patients with tuberous sclerosis (TS)

Much greater amounts of small polydisperse circular DNA (spcDNA) have been detected, in cell cultures derived from angiofibromas of six patients with tuberous sclerosis (TS) than in those from the skin of these patients or from the skin of 11 healthy donors. This observation could be confirmed by spreading the DNA of appropriate fractions from CsCl density gradients. The findings suggest the existence of a relationship between the chromosomal instability observed in angiofibroma cultures and the mobilization of spcDNA.

Recognition of covalent insulin-receptor complexes on viable adipocytes by anti-insulin antibodies.

Isolated rat adipocytes were photo-affinity-labelled with B2-(4-azido-2-nitrophenylacetyl)-des-PheB1-insulin or B29-(4-azido-2-nitrophenylacetyl)insulin. Four anti-insulin antibodies (3 monoclonal, 1 polyclonal) were tested for their ability to inhibit the persistent stimulation of lipogenesis caused by the covalently bound insulin [Brandenburg et al. (1980) Nature (London) 286, 821-822]. The polyclonal and 2 monoclonal antibodies, directed against the C-terminus of the B-chain, gave a significant depression, while one antibody, directed against the region A(8-10), was without effect. Under reversible conditions, without irradiation, all antibodies completely inhibited lipogenesis. For the polyclonal antibody this is shown in a dose-dependent way. It is concluded that the effective antibodies can recognize their epitope because it is accessible on the surface of the complex and does not represent part of the receptor-binding surface of insulin. This binding leads to interference with the generation and/or transmittance of the biological signal.

Structure of viroid replicative intermediates: physico-chemical studies on SP6 transcripts of cloned oligomeric potato spindle tuber viroid.

The structure and structural transitions of transcripts of cloned oligomeric viroid were studied in physico-chemical experiments and stability calculations. Transcripts of (+) and (-) polarity, from unit up to sixfold length, were synthesized from DNA clones of the potato spindle tuber viroid (PSTV) with the SP6 transcription system. Their structural properties were investigated by optical denaturation curves, high performance liquid chromatography (HPLC), electron microscopy, sedimentation-diffusion equilibrium and velocity sedimentation. Secondary structures of the RNAs and theoretical denaturation curves were calculated using an energy optimization program. The secondary structure of lowest free energy for unit length and oligomeric transcripts is a rod-like structure similar to that of the mature circular viroids. When this structure is used as a model for calculations, there is a large degree of agreement between the theoretical and the experimental denaturation curves. At high temperatures, however, (+) strand transcripts exhibited a transition which was more stable than expected from the calculations or than was known from curves of mature viroids. This transition arises from a rearrangement of the central conserved region of viroids to a helical region of 28 stable base pairs either intermolecularly leading to bimolecular complexes, or intramolecularly giving rise to a branched secondary structure. The rearrangement could be detected by electron microscopy, HPLC, and analytical ultracentrifugation. The helical region serves to divide up the oligomeric (+) strand into structural units which may be recognized by cleavage and ligation enzymes which process the oligomeric intermediates to circular mature viroids.