Thierry Lambert

In vivo transfer of genetic information between gram-positive and gram-negative bacteria.

A 1427‐bp DNA fragment containing the kanamycin resistance gene, aphA‐3, of plasmid pIP1433 from Campylobacter coli was inserted into a shuttle vector. Full expression of aphA‐3 was obtained in Bacillus subtilis and in Escherichia coli. This DNA fragment was sequenced in its entirety and the starting point for aphA‐3 transcription in B. subtilis, C. coli and E. coli was determined by S1 nuclease mapping. The sequence of the promoter consists of the hexanucleotides TTGACA and TATAAT, with a spacing of 17 bp. The nucleotide sequence of the aphA‐3 gene from C. coli and from the streptococcal plasmid pJH1 are identical whereas they differ by two substitutions and deletion of a codon from that cloned from the staphylococcal plasmid pSH2. These results indicate a recent extension of the resistant gene pool of Gram‐positive cocci to Gram‐negative bacilli. From an analysis of the DNA sequences surrounding the promoter region, we concluded that the DNA fragment containing the aphA‐3 gene in plasmid pJH1 has evolved by deletions from a sequence similar to that found in plasmid pIP1433.

Structural relationship between the genes encoding 3′-aminoglycoside phosphotransferases in Campylobacter and in gram-positive cocci

Campylobacter coli strain BM2509 resistant to ampicillin, chloramphenicol, erythromycin, kanamycin, spectinomycin, streptomycin and tetracycline was isolated from a patient with hospital-acquired diarrhoea. Resistance to kanamycin has not been thus far described in Campylobacter. Phosphocellulose paper-binding assay indicated that resistance to kanamycin and structurally related antibiotics in strain BM2509 was due to synthesis of a 3-aminoglycoside phosphotransferase of type III (APH(3)-III), an enzyme so far confined to Gram-positive cocci. The kanamycin and tetracycline resistances were transferable en bloc by conjugation to C. fetus but not to Escherichia coli. Analysis by agarose gel electrophoresis of crude bacterial lysates revealed the presence, in BM2509 and in the transconjugants, of a plasmid, pIP1433, with a size of 47.2 kilobases (Kb). Strain BM2509 also harboured a 4.5-Kb cryptic plasmid. DNA annealing studies indicated a close structural relationship between the kanamycin resistance gene of C. coli BM2509 and that representative of this type of resistance determinant in Gram-positive cocci. These results indicate that emergence of resistance to kanamycin in Campylobacter is due to acquisition in vivo of a gene or a plasmid from Gram-positive bacteria.

Native Valve Endocarditis Due to Enterococcus hirae

ABSTRACT Enterococcus hirae is a rare isolate in clinical specimens. We describe a case of native aortic-valve endocarditis that was caused by Enterococcus hirae in a 72-year-old man. This is the first reported case of endocarditis due to this organism.

Protein A Sepharose immunoadsorption can restore the efficacy of platelet concentrates in patients with Glanzmann's thrombasthenia and anti-glycoprotein IIb-IIIa antibodies

Summary. Type I Glanzmanns thrombasthenia is a rare congenital platelet function disorder, characterized by undetectable platelet membrane glycoprotein IIb–IIIa (GPIIb–IIIa). Severe bleeding is controlled by transfusion of normal platelets, leading in some cases to the occurrence of anti‐GPIIb–IIIa isoantibodies, which induces a loss of transfused platelet efficacy. We used immunoadsorption on protein A Sepharose (IA‐PA), which has been shown to be efficient in decreasing the titre of antibodies in several immune diseases, in three patients with Glanzmanns thrombasthenia and anti‐GPIIb–IIIa isoantibodies on five different occasions. IA‐PA was well tolerated with no deleterious side‐effects reported. It induced a dramatic decrease of total immunoglobulin (Ig)G, including anti‐GPIIb–IIIa isoantibody levels, as assessed by the monoclonal antibody‐specific immobilization of platelet antigens test and the ex vivo inhibition of normal platelet aggregation induced by the patients platelet‐rich or platelet‐poor plasma. Elimination of the antibody was associated with a correction of the bleeding time following platelet transfusion. IA‐PA combined with platelet transfusion made it possible to control two life‐threatening haemorrhages, and allowed two surgical procedures and one bone marrow transplantation to be performed safely. Our experience suggests that IA‐PA, which restores the haemostatic efficacy of platelet transfusion, is a valuable therapeutic strategy in patients with Glanzmanns thrombasthenia and anti‐GPIIb–IIIa isoantibodies.

Protein A sepharose immunoadsorption: immunological and haemostatic effects in two cases of acquired haemophilia

Acquired haemophilia is a life‐threatening disorder caused by circulating auto‐antibodies that inhibit factor VIII coagulant activity (FBIII:C). Immunoadsorption on protein A sepharose (IA‐PA) was performed in two bleeding patients with acquired haemophilia: we observed a dramatic and quick decrease in the anti‐FVIII:C inhibitor titre leading to a normal, albeit transient, haemostatic status. In one case, IA‐PA was the only procedure which succeeded in stopping massive haemorrhage. In the second case, IA‐PA reinforced the haemostatic effect of recombinant activated factor VII by increasing the endogenous plasma factor VIII level. The efficacy of IA‐PA was sustained with immunosuppressive treatment introduced, respectively, 10 and 15u2003d before the IA‐PA procedures. Our experience with IA‐PA suggests that this extracorporeal anti‐FVIII:C removal procedure is a valuable therapeutic tool for acquired haemophilia and can alleviate life‐threatening haemorrhages.

Injection of recombinant activated factor VII can induce transient increase in circulating procoagulant microparticles

Recombinant activated factor VII (rFVIIa) is an effective haemostatic treatment in haemophiliacs with inhibitors. In vitro, FVIIa concentrations corresponding to those obtained with therapeutic doses of rFVIIa have been shown to induce normal thrombin generation and platelet activation in the absence of factors VIII or IX. To further study the in vivo haemostatic changes induced by rFVIIa, circulating procoagulant microparticles (MP) were measured in patients treated with discontinuous injections of Novoseven. In 6 out of 15 patients, a transient peak of procoagulant MP was observed after injection, occurring 15 min to 2 h after infusion. It was composed primarily of platelet-derived MP and was of very short duration. This peak was not observed in haemophiliacs without inhibitor, who were treated with conventional replacement therapies. Our results provide further in vivo evidence that rFVIIa specifically activates platelets, either directly or as a consequence of a burst of thrombin generation that could account for its haemostatic efficacy.

Reappraisal of in utero Stem Cell Transplantation Based on Long-Term Results

The therapeutic field of in utero transplantation of stem cells, into human fetuses, has developed since 1988 with the hope of improved probability of engraftment and tolerance, due to immune immaturity of the host. Fifteen years later, it is possible to evaluate the results that we and others have obtained in the treatment of several fetal diseases. Seven fetal patients have been treated in Lyon: In 2 cases, pregnancy termination was induced by the in utero injection; in the 5 other cases, engraftment was obtained and repeatedly documented with presence of donor HLA antigens and/or Y chromosome in recipients. In the 2 patients with combined immunodeficiency disease, a sustained reconstitution of immunity was obtained as a result of the transplant but other complications occurred thereafter. In patients with thalassemia major, Niemann-Pick disease or hemophilia, a very partial and very transitory benefit was only obtained. Approximately 33 other patients with immunodeficiencies, hemoglobinopathies or inborn errors of metabolism have been treated worldwide, over the last 13 years, with a comparable method, using parental or fetal stem cells transplanted in utero. Successful treatment has usually been recorded in immunodeficiencies, and insufficient results have been obtained in the other cases. This form of treatment can therefore be recommended after prenatal diagnosis of combined immunodeficiency but additional research is required to improve the degree of engraftment, the lack of resistance of the host and the ‘space’ available for hematopoiesis in the other conditions.

Analysis of the Mobilization Functions of the Vancomycin Resistance Transposon Tn1549, a Member of a New Family of Conjugative Elements

Conjugative transfer from Clostridium symbiosum to enterococci of Tn1549, which confers VanB-type vancomycin resistance, has been reported. This indicates the presence of a transfer origin (oriT) in the element. Transcription analysis of Tn1549 indicated that orf29, orf28, orfz, and orf27 were cotranscribed. A pACYC184 derivative containing 250 bp intergenic to orf29-orf30 of Tn1549 was mobilized in Escherichia coli recA::RP4::Delta nic provided that orf28 and orf29 were delivered simultaneously. These open reading frame (ORF) genes were able to promote mobilization in trans, but a cis-acting preference was observed. On the basis of a mobilization assay, a minimal 28-bp oriT was delimited, although the frequency of transfer was significantly reduced compared to that of a 130-bp oriT fragment. The minimal oriT contained an inverted repeat and a core, which was homologous to the cleavage sequence found in certain Gram-positive rolling-circle replicating (RCR) plasmids. While Orf29 was a mobilization accessory component similar to MobC proteins, Orf28 was identified as a relaxase belonging to a new phyletic cluster of the MOB(p) superfamily. The nick site was identified within oriT by an oligonucleotide cleavage assay. Closely related oriTs linked to mobilization genes were detected in data banks; they were found in various integrative and conjugative elements (ICEs) originating mainly from anaerobes. These results support the notion that Tn1549 is a member of a MOB(p) clade. Interestingly, the Tn1549-derived constructs were mobilized by RP4 in E. coli, suggesting that a relaxosome resulting from DNA cleavage by Orf28 interacted with the coupling protein TraG. This demonstrates the capacity of Tn1549 to be mobilized by a heterologous transfer system.

Distribution of the vanG-like gene cluster in Clostridium difficile clinical isolates

Treatment of Clostridium difficile infections generally requires cessation of their causative antibiotic and subsequent administration of metronidazole or vancomycin. Intriguingly, the genome of C. difficile 630 contains a cryptic gene cluster homologous to the vanG-type operon of Enterococcus faecalis BM4518. We detected this cluster by PCR in 35 out of 41 clinical isolates, confirming its large prevalence in this species. The cluster was found to be located in a unique locus. Comparison of this locus with that of strains devoid of the vanG-like cluster indicated that acquisition of the gene cluster occurred in a perfect 19-bp inverted repeat, in the absence of a detectable mobile structure.

Clostridium clostridioforme and Atopobium minutum Clinical Isolates with VanB-Type Resistance in France

ABSTRACT Acquired vancomycin resistance in Gram-positive anaerobes has been reported only in Australia and Canada from rare vanB-positive stool samples in the absence of vancomycin-resistant enterococci (VRE). We report the emergence of VanB-type resistance in Clostridium clostridioforme and Atopobium minutum involved in human infections in France.