[Multifactorial analysis of the phenotypes for beta-lactams of 1044 Escherichia coli strains]. / Analyse multifactorielle des phénotypes aux bêtalactamines de 1044 souches de Escherichia coli.

1,044 E. coli strains were randomly collected by the beginning of 1992. Their susceptibility for seven beta-lactam antibiotics: amoxycillin, augmentin, ticarcillin, claventin, cephalothin, cefoxitin and cefotaxime, was studied routinely by the agar diffusion method. The datas were analyzed by the CERIB multifactorial analysis package which yields to homogeneous populations. This analysis showed four well defined populations: 1) 588 strains (56.4%) susceptible to all antibiotics; 2) 410 strains (39.3%) present a penicillinase phenotype; 3) 11 strains (1.05%) are cephalosporinase producer; 4) 7 strains (0.67%) were identified as producing an extended-spectrum beta-lactamase. The remaining strains: 28 (2.68%) had a reduced susceptibility to all antibiotics, which suggests the combination of few resistance mechanisms or other hypothesis.

Sequence requirements for target activity in site-specific recombination mediated by the Int protein of transposon Tn 1545.

Excision and integration of Tn1545 occur by reciprocal site-specific recombination between 6 (or 7) bp variable sequences present in the recombining attachment (att) sites and designated overlap regions. We devised an assay for Tn1545 transposition in which derivatives containing the cis-acting transposition sequences (attTn 1545) integrate into a target replicon when complemented in trans by the transposon-encoded integrase Int-Tn. This assay was used to determine the characteristics of the DNA sequence that influence target site selection. Characterization of several integration sites indicated that a 20 bp segment, designated attB, contains the sequences required for target activity. It also appeared that (i) the target activity depends upon the extent of homology between the 7bp segments flanking the overlap regions in attB and attTn 1545, and (ii) the degree of homology between the two recombining overlap regions does not affect the level of target activity and has no influence on integration orientation.

Genetic basis of tetracycline resistance in clinical isolates of Listeria monocytogenes.

The genetic basis of tetracycline resistance was studied in 25 clinical isolates of Listeria monocytogenes. Resistance to tetracycline was associated with resistance to minocycline and due to the presence of the tet(M) gene in 24 strains. Association of tet(M) with int-Tn, the gene encoding the protein required for the movements of Tn1545-like conjugative transposons, was found in all strains. Cotransfer of tet(M) and int-Tn among L. monocytogenes cells and from L. monocytogenes to Enterococcus faecalis was detected in 7 of the 12 strains studied at frequencies similar to those obtained with the prototype element Tn1545. tet(L), the second most prevalent tetracycline resistance gene in enterococci and streptococci, was detected in the remaining strain, where it was borne by a 5-kb plasmid. These observations indicate that two types of movable genetic elements, transposons and plasmids, in enterococci and streptococci are responsible for emergence of drug resistance in L. monocytogenes.

An integrative vector exploiting the transposition properties of Tn1545 for insertional mutagenesis and cloning of genes from gram-positive bacteria.

We have constructed and used an integrative vector, pAT112, that takes advantage of the transposition properties (integration and excision) of transposon Tn1545. This 4.9-kb plasmid is composed of: (i) the replication origin of pACYC184; (ii) the attachment site (att) of Tn1545; (iii) erythromycin-and kanamycin-resistance-encoding genes for selection in Gram- and Gram+ bacteria; and (iv) the transfer origin of IncP plasmid RK2, which allows mobilization of the vector from Escherichia coli to various Gram+ recipients. Integration of pAT112 requires the presence of the transposon-encoded integrase, Int-Tn, in the new host. This vector retains the insertion specificity of the parental element Tn1545 and utilises it to carry out insertional mutagenesis, as evaluated in Enterococcus faecalis. Since pAT112 contains the pACYC184 replicon and lacks most of the restriction sites that are commonly used for molecular cloning, a gene from a Gram+ bacterium disrupted with this vector can be recovered in E. coli by cleavage of genomic DNA, intramolecular ligation and transformation. Regeneration of the gene, by excision of pAT112, can be obtained in an E. coli strain expressing the excisionase and integrase of Tn1545. The functionality of this system was illustrated by characterization of an IS30-like structure in the chromosome of En. faecalis. Derivatives pAT113 and pAT114 contain ten unique cloning sites that allow screening of recombinants having DNA inserts by alpha-complementation in E. coli carrying the delta M15 deletion of lacZ alpha. These vectors are useful to clone and introduce foreign genes into the genomes of Gram+ bacteria.

Nucleotide sequences specific for Tn1545-like conjugative transposons in pneumococci and staphylococci resistant to tetracycline.

The distributions of tet(M) and conjugative transposons related to Tn1545 were studied by hybridization in 47 clinical isolates of Streptococcus pneumoniae resistant to tetracycline. Resistance to tetracycline was always associated with resistance to minocycline and was due to the presence of the tet(M) gene. Association of tet(M) with int-Tn, the gene encoding the protein required for the movements of Tn1545-like transposons, was found in all but one strain of S. pneumoniae. In contrast, int-Tn was detected in only 2 of 37 strains of Staphylococcus spp. harboring tet(M).

Conjugative transposition of Tn916 requires the excisive and integrative activities of the transposon-encoded integrase.

Transposon Tn916 is a 16.4-kb broad-host-range conjugative transposon originally detected in the chromosome of Enterococcus faecalis DS16. Transposition of Tn916 and related transposons involves excision of a free, nonreplicative, covalently closed circular intermediate that is substrate for integration. Excisive recombination requires two transposon-encoded proteins, Xis-Tn and Int-Tn, whereas the latter protein alone is sufficient for integration. Here we report that conjugative transposition of Tn916 requires the presence of a functional integrase in both donor and recipient strains. We have constructed a mutant, designated Tn916-int1, by replacing the gene directing synthesis of Int-Tn by an allele inactivated in vitro. In mating experiments, transfer of Tn916-int1 from Bacillus subtilis to E. faecalis was detected only when the transposon-encoded integrase was supplied by trans-complementation in both the donor and the recipient. These results suggest that conjugative transposition of Tn916 requires circularization of the element in the donor followed by transfer and integration of the nonreplicative intermediate in the recipient.

Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria.

The mobilizable shuttle cloning vectors, pAT18 and pAT19, are composed of: (i) the replication origins of pUC and of the broad-host-range enterococcal plasmid pAM beta 1; (ii) an erythromycin-resistance-encoding gene expressed in Gram- and Gram+ bacteria; (iii) the transfer origin of the IncP plasmid RK2; and (iv) the multiple cloning site and the lacZ alpha reporter gene of pUC18 (pAT18) and pUC19 (pAT19). These 6.6-kb plasmids contain ten unique cloning sites that allow screening of derivatives containing DNA inserts by alpha-complementation in Escherichia coli carrying the lacZ delta M15 deletion, and can be efficiently mobilized by self-transferable IncP plasmids co-resident in the E. coli donors. Plasmids pAT18, pAT19 and recombinant derivatives have been successfully transferred by conjugation from E. coli to Bacillus subtilis, Bacillus thuringiensis, Listeria monocytogenes, Enterococcus faecalis, Lactococcus lactis, and Staphylococcus aureus at frequencies ranging from 10(-6) to 10(-9). The presence of a restriction system in the recipient dramatically affects (by three orders of magnitude) the efficiency of conjugal transfer of these vectors from E. coli to Gram+ bacteria.

The integration-excision system of the conjugative transposon Tn 1545 is structurally and functionally related to those of lambdoid phages.

Excision of Tn1545 and related conjugative transposons of Gram-positive bacteria occurs by reciprocal site-specific recombination between non-homologous regions of the transposon-target junctions. Excisive recombination requires two transposon-encoded proteins designated Xis-Tn and Int-Tn. We have shown that, following excision, Tn1545 is a circular structure with ends separated by either of the two hexanucleotides that were present at the transposon-target junctions. Using a trans-complementation assay, we have demonstrated that Int-Tn is able to catalyse in vivo integration of a circular intermediate of Tn1545 defective for integration and excision. comparison of integration sites suggests that limited sequence homology at the vicinity of the recombining sites is required for integration of the element. These data support the hypothesis that the integration/excision systems of conjugative transposons from Gram-positive cocci and of lambdoid phages from Gram-negative bacilli have evolved from a common ancestor.

Transferable plasmid-mediated antibiotic resistance in Listeria monocytogenes.

A strain of Listeria monocytogenes, isolated from a patient with meningoencephalitis, was resistant to chloramphenicol, erythromycin, streptomycin, and tetracycline. The genes conferring resistance to these antibiotics were carried by a 37-kb plasmid, pIP811, that was self-transferable to other L monocytogenes cells, to enterococci-streptococci, and to Staphylococcus aureus. The efficacy of transfer and the stability of pIP811 were higher in enterococci-streptococci than in the other gram-positive bacteria. As indicated by nucleic acid hybridisation, the genes in pIP811 conferring resistance to chloramphenicol, erythromycin, and streptomycin were closely related to plasmid-borne determinants that are common in enterococci-streptococci. Plasmid pIP811 shared extensive sequence homology with pAM beta 1, the prototype broad host range resistance plasmid in these two groups of gram-positive cocci. These results suggest that emergence of multiple antibiotic resistance in Listeria spp is due to acquisition of a replicon originating in enterococci-streptococci. The dissemination of resistance to other strains of L monocytogenes is likely.

Antibacterial and plasmid curing activity of lomefloxacin in vitro.

The in vitro activity of lomefloxacin in comparison to other recently developed quinolones was evaluated by determination of MICs for 89 test strains. Organisms tested included clinical isolates of gram-positive and gram-negative bacteria susceptible or resistant to quinolones, resistant mutants of Enterobacteriaceae, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis obtained in vitro, and the wild type strain Escherichia coli K12 with its mutants na1A, nalB, nalC and nalB. The activity of lomefloxacin was similar to that of pefloxacin against gram-positive cocci and similar to that of norfloxacin against gram-negative bacteria. There was cross-resistance to all quinolones tested in pefloxacin resistant clinical isolates and in laboratory mutants. The activity of lomefloxacin was decreased against the Escherichia coli K12 mutants nalA and nalD but not nalB, whereas the nalC mutant showed increased susceptibility. Susceptible and mutant strains (n = 115) were used to establish the least-squares lines of regression with 5-micrograms and 10-micrograms lomefloxacin disks. Tests of the in vitro plasmid curing activity of lomefloxacin compared to other agents showed no statistically significant plasmid loss after treatment with lomefloxacin.

Molecular characterization of two proteins involved in the excision of the conjugative transposon Tn1545: homologies with other site-specific recombinases.

Excision is probably the initial and rate-limiting step of the movements of conjugative transposons of Gram-positive bacteria such as Tn916 and Tn1545. We have shown, by molecular cloning and DNA sequencing, that a 2058 bp Sau3A right-junction fragment of transposon Tn1545 specifies two gene products that are involved in the excision of the element. The DNA sequence of these genes, designated orf1 and orf2, has been determined and the corresponding proteins, ORF1 and ORF2, have been identified in a bacterial cell-free coupled transcription-translation system. These proteins are freely diffusible since they are able to trans-complement in vivo a deletion derivative of Tn1545 defective for excision. Using an in vivo complementation assay, we have demonstrated that ORF2 alone is able to catalyse excision and that ORF1 strongly stimulates the activity of ORF2. We also found that ORF1 and ORF2 display local homology with, respectively, proteins Xis and Int from lamboid phages, which suggests that these excision systems have a common origin. Based on the functional properties of the integrase of bacteriophage lambda, on the analysis of the nucleotide sequence of the junction fragments and of the target before insertion and after excision, a model is proposed for ORF2-catalysed excision of Tn1545 and related conjugative transposons.

[Effect of various antibiotics on granulopoiesis, in mice treated with cyclophosphamide, in an in vivo model of experimental C. albicans infection]. / Effects de différents antibiotiques sur la granulopoïèse, chez la souris traitée par cyclophosphamide, dans un modèle in vivo d'infection expérimentale par C. albicans.

The effect of piperacillin (PIP), cefotaxime (CTX), cefoxitin (CXT) on the natural resistance to C. albicans infection has been evaluated in vivo, in normal or neutropenic mice, in correlation with the PMN count in the peripheral blood. In neutropenic mice treated with PIP or CTX, the number of PNN increased more rapidly and higher than in CXT treated or control mice. A dose dependent increased resistance to infection was observed in PIP treated mice. It did not parallelled the PMN level found in different group of mice. The explanations might be a decrease of functional activity of PMN and/or an increase proliferation and differentiation of others effector cytotoxic cells.