Blockage of the urokinase receptor on the cell surface: construction and characterization of a hybrid protein consisting of the N-terminal fragment of human urokinase and human albumin.

Receptor-bound urokinase is likely to be a crucial determinant in both tumor invasion and angiogenesis. We report here that a yeast-derived genetic conjugate between human serum albumin and the 1-135 N-terminal residues of urokinase (u-PA) competitively inhibits the binding of exogenous and endogenous u-PA to its cell-anchored receptor (u-PAR). This hybrid molecule (ATF-HSA) also inhibits in vitro pro-urokinase-dependent plasminogen activation in the presence of u-PAR bearing cells. These effects are probably responsible for the observed in vitro inhibition of tumor cell invasion in a reconstituted basement membrane extract (Matrigel).

Blockage of urokinase receptor reduces in vitro the motility and the deformability of endothelial cells.

The binding of urokinase (u-PA) to its cell surface receptor (u-PAR) is critical for tumor cell invasion. Here, we report that the distribution of this binding by a u-PAR antagonist ATF-HSA inhibits in vitro the motility of endothelial cells in a dose-dependent manner. This inhibition was also observed when the cells were first stimulated with potent angiogenic factors, including bFGF or VEGF. [3H]thymidine incorporation assay demonstrated that ATF-HSA did not affect the cell proliferation. ATF-HSA was more potent than plasmin inhibitors, suggesting that it exerts its effects not solely by inhibiting the remodeling of the extracellular matrix. In fact, analysis of the cell shape change during migration revealed for the first time that its effect is related to a decrease in cell deformability. These results suggest that u-PAR antagonist may be a new approach to control angiogenesis.

Molecular epidemiology of integron-associated antibiotic resistance genes in clinical isolates of enterobacteriaceae.

The epidemiology of integron-mediated antibiotic-resistant genes in clinical enterobacteria from a single location was investigated. Forty-nine isolates (kindly provided by Dr. D. Sirot, Clermont-Ferrand, France) were selected for transferable resistance to aminoglycosides or to other antibiotics. Total DNA prepared from these strains was screened for the presence of conserved segments of integrons by PCR. The nature and frequency of inserted resistance gene cassettes were determined by direct nucleotide sequencing and were related to the resistances expressed by the strain. Integron hot-spots were present in 59% of the strains from 6 species, in either one or two copies. For amplicons sequenced, one or two antibiotic-resistant genes were found in various combinations, and were always expressed at the phenotypic level. They included the aminoglycoside resistance genes ant(3")-Ia and aac(6')-Ib (75%), as well as dhfr-I,-VII (21.4%) and blaOXA-1 (3.6%). Almost half of the transferable resistance to aminoglycosides (53%) was mediated by integron hot-spots in strains characterized at the nucleotide level. The proportion rose to 100% for the AAC(6')-I resistance profile. This study emphasizes the important contribution of integrons to aminoglycoside resistance within enterobacteria from a clinical setting.

Biotinylated oligonucleotide probes for the detection and the characterization of TEM-type extended broad spectrum beta-lactamases in Enterobacteriaceae.

Point mutations in the nucleotide sequence of the structural genes for the TEM-type penicillinases can broaded their substrate spectrum towards all beta-lactams except cephamycins and imipenem. The presence of such variants on self-transferable plasmids accounts for the dissemination of this new type of resistance to numerous species of Enterobacteriaceae in various countries. We have synthetized biotinylated oligonucleotide probes for the detection and the discrimination of parental and mutated nucleotide sequences of TEM enzymes. Seven clinical isolates belonging to four species and harbouring TEM-1, TEM-3 or TEM-6 were studied. The results obtained indicate that detection of TEM-derived broad spectrum beta-lactamases in clinical isolates of Entero-bacteriaceae is possible with biotinylated oligonucleotide probes.

Two novel transferable extended-spectrum beta-lactamases from Klebsiella pneumoniae in Tunisia.

Two novel beta-lactamases conferring multiresistance to antibiotics including oxyimino beta-lactams have been identified in two nosocomial K. pneumoniae strains isolated in Tunis in 1986 and 1988. Both enzymes were encoded by ca. 150-kilobase plasmids. Donor and transconjugant strains producing these enzymes exhibited highly similar pattern of resistance (CTX phenotype) to beta-lactams including penicillins and oxyimino beta-lactams e.g. cefotaxime, ceftriaxone, ceftazidime, and aztreonam. High and variable synergy (16 to 1066-fold) was obtained when combined to 0.1 microgram/ml of clavulanate (beta-lactamase inhibitor). The isoelectric points of these two enzymes were 5.4 and 6.4. These beta-lactamases differed from TEM types by hydrolysis for cefotaxime or ceftriaxone but were inhibited by clavulanate and cloxacillin. DNA hybridization studies suggested that that the genes of these enzymes may be derived from genes encoding TEM-type enzymes.

Two variants of transferrable extended-spectrum TEM-beta-lactamase successively isolated from a clinical Escherichia coli isolate.

In a leukaemic patient presenting a septicaemia treated with ceftazidime and amikacin, two clinical Escherichia coli isolates distinguished by their level of resistance to oxyimino-beta-lactams were isolated at an interval of 24 h. The isolates were identified by biotyping and esterase electrophoretic typing and the two host strains were shown to be identical. However, each of these strains exhibited a different transferrable extended-spectrum beta-lactamase. These enzymes had different pI values (5.25 and 5.58), but were both blaTEM-1 mutants. The enzyme with pI 5.25 was identical to TEM-101 (TEM-12) (serine 162 substitution). The enzyme with pI 5.58 showed an additional amino acid substitution (lysine residue instead of an arginine at position 237) and was denominated TEM-23. These data indicate that point-mutations can be successively cumulated in vivo by blaTEM mutants, leading to expression of beta-lactamases with increased hydrolysis rates.

Automated RNA probe assay for the identification of Listeria monocytogenes.

Recent epidemics of human listeriosis have shown the importance of Listeria monocytogenes as a food-borne risk. We have developed an automated identification assay for L. monocytogenes using the specificity of the 16S rDNA probe described by Wang et al. (1991). Bacterial cultures were lysed quickly by a chemical step releasing the target nucleic acids. Extracts were loaded into the VIDAS automate (bioMérieux) which performed a non-radio-active hybridisation reaction for 2 h. This assay recognised specifically 68 out of 69 L. monocytogenes isolates from clinical and food origins, including serovars 4b and 1/2, without cross-hybridising to other Listeria species (103 strains) or other bacterial species (15 strains). The sensitivity of the assay was 10(7) bacteria. This automated technology is faster than conventional biochemical identification.

Development of "oligotyping" for characterization and molecular epidemiology of TEM beta-lactamases in members of the family Enterobacteriaceae.

Based on the DNA sequences of blaTEM-1 and blaTEM-2, which encode parental penicillinases TEM-1 and TEM-2, respectively, and blaTEM-3, blaTEM-4, blaTEM-5, blaTEM-6, and blaTEM-7, which encode extended-spectrum beta-lactamases, we designed heptadecanucleotides to discriminate point mutations in five loci. Determination of the hybridization profiles by colony hybridization with this selection of probes, termed "oligotyping," allowed characterization of the TEM variants present in 265 clinical isolates of the family Enterobacteriaceae that exhibit synergism between a penicillinase inhibitor and broad-spectrum cephaslosporins. Among the 222 strains harboring TEM enzymes, Klebsiella pneumoniae (48%) and Escherichia coli (21%) were predominant, and TEM-3 was the most common enzyme (60%). Penicillinases TEM-1 and TEM-2 were detected alone (15 and 1%, respectively), combined (1%), or associated with another TEM beta-lactamase (17 and 6%, respectively). Fourteen variants, including seven new enzymes, were detected. One, TEM-13, was a new penicillinase with the same isoelectric point and substrate range as TEM-2 but differed by a single amino acid substitution, whereas the others, TEM-14 to TEM-19, were extended-spectrum beta-lactamases that consisted of novel combinations of known amino acid substitutions. Different TEM variants were found to coexist within the same cells. A patient could harbor two or three different strains that encoded the same enzyme or two indistinguishable isolates that produced distinct TEM beta-lactamases.

Emergence of aminoglycoside resistance genes aadA and aadE in the genus Campylobacter.

Resistance to streptomycin or spectinomycin or both in five Campylobacter coli strains, two Campylobacter jejuni strains, and a Campylobacter-like strain was studied by enzymatic assays and dot blot hybridization. Resistance was due to 6- or 3",9-aminoglycoside adenylyltransferases and to new types of phospho- and adenylyltransferases.

Detection of erythromycin resistance by the polymerase chain reaction using primers in conserved regions of erm rRNA methylase genes.

Genes belonging to different erm DNA hybridization classes were selectively amplified by polymerase chain reaction with a pair of oligonucleotides that corresponded to conserved amino acid motifs in known ERM methylases. Identification of the resistance mechanism was possible despite substantial nucleotide sequence diversity among the erythromycin resistance genes.

A new example of physical linkage between Tn1 and Tn21: the antibiotic multiple-resistance region of plasmid pCFF04 encoding extended-spectrum beta-lactamase TEM-3.

The genetic environment of plasmid-borne blaTEM mutant genes, encoding nine distinct TEM-type extended-spectrum beta-lactamases, was studied in transconjugants from clinical isolates of enterobacteria. Colony hybridization with probes specific for tnpA and tnpR of Tn3, tnpA and tnpI of Tn21, aacA4, and IS15, and restriction endonuclease analysis of plasmid DNA indicated that the structural genes for the enzymes were always associated with intact or deleted variants of the Tn3 family. Four of the nine blaTEM variants, which account for 62% of 222 isolates in a molecular epidemiological study, were associated with replicons indistinguishable from the epidemic Inc7-M plasmid pCFF04 that carries the blaTEM-3 gene. This suggests that mutant genes were selected from the same prototype plasmid carrying penicillinase genes blaTEM-1 or -2. A 6.6 kb DNA fragment of pCFF04 containing blaTEM-3 was characterized by amplification mapping and sequencing. The results obtained indicated that blaTEM-3 was present on a copy of Tn1 interrupted at the start codon of the transposase by a DNA sequence reminiscent of the inverted repeats of class II transposons. This partial Tn1 copy was in turn, inserted into the transposase gene of a Tn21-like transposon containing an integron expressing an aacA4 gene. The presence of an integron can account for the various assortments of aminoglycoside resistance genes found associated with blaTEM-3.

Direct sequencing of the amplified structural gene and promoter for the extended-broad-spectrum beta-lactamase TEM-9 (RHH-1) of Klebsiella pneumoniae.

Extended-broad-spectrum beta-lactamase TEM-9, detected in a clinical isolate of Klebsiella pneumoniae, confers high-level resistance to recent cephalosporins, in particular ceftazidime, and to the monobactam aztreonam. Using oligonucleotide probes, we found that the plasmid gene blaT-9 encoding TEM-9 differs from characterized blaT genes by a new combination of already known mutations. Gene blaT-9 was further studied by direct sequencing of an amplified 1.1-kb DNA fragment which contained the open reading frame and its promoter. Analysis of the nucleotide and of the deduced amino acid sequence confirmed the hybridization results and indicated that TEM-9 differs from TEM-1 by four amino acid substitutions: Phe at position 19 and Met at position 261, which have been found in TEM-4 and are known not to expand the enzyme substrate range; Lys 102, detected in TEM-3 and TEM-4, and Ser 162, present in TEM-5 and TEM-7. Each of the latter substitutions enlarges the substrate spectrum of the enzymes and they are found associated for the first time in TEM-9.

An IS1-like element is responsible for high-level synthesis of extended-spectrum beta-lactamase TEM-6 in Enterobacteriaceae.

Resistance of Escherichia coli strain HB251 to the newer beta-lactam antibiotics, in particular ceftazidime and aztreonam, results from production of the extended-spectrum beta-lactamase TEM-6. The corresponding structural gene, bla(T)-6, and its promoter region were amplified by the polymerase chain reaction. Analysis of the sequence of the amplification product showed that bla(T)-6 differed by two nucleotide substitutions from bla(T)-1, the gene encoding TEM-1 penicillinase in plasmid pBR322. The mutations led to the substitution of a lysine for a glutamic acid at position 102 and of a histidine for an arginine at position 162 of the unprocessed TEM-1 protein. The presence of a 116 bp DNA insert upstream from bla(T)-6 resulted in the creation of hybrid promoter P6 in which the -10 region was that of TEM-1 promoter P3 whereas the -35 canonical sequence TTGACA was provided by the right end of the insert. P6 was found to be 10 times more active than P3 and to confer higher levels of antibiotic resistance upon the host. Analysis of the sequence of the insert indicated that the 116 bp fragment is related to insertion sequence IS1 but differs from it by three internal deletions that removed regions encoding the transposase. The distribution of the IS1-like element in clinical isolates of Enterobacteriaceae was studied by the polymerase chain reaction and by DNA-DNA hybridization. The element appeared to be widespread and was detected in strains producing TEM-6 or other TEM variants.

[Relationship between the level of minimal inhibitory concentrations of five antiseptics and the presence of qacA gene in Staphylococcus aureus]. / Relation entre le niveau des concentrations minimales inhibitrices de cinq antiseptiques et la présence du gène qacA chez Staphylococcus aureus.

The Polymerase Chain Reaction (PCR) was used to detect the qacA gene which encodes antiseptic resistance in 186 clinical isolates of Staphylococcus aureus. The results were compared with those obtained by MIC determination of 4 antiseptics (benzalkonium chloride, hexamidine, chlorhexidine, acriflavine) and for ethidium bromide. The qacA gene was not detected among the 32 susceptible S. aureus strains, but was found in the 70 (85%) of the 82 S. aureus strains resistant to all 5 antiseptics. The gene was also detected in 70 (45%) of the 154 remaining strains that were resistant to at least one antiseptic.

Comparative analysis of 16S and 23S rRNA sequences of Listeria species.

In order to establish the taxonomic value of 16S rRNA and 23S rRNA for distinguishing Listeria species, the complete 23S rRNA sequences for all Listeria species were determined by using the type strains. We designed and experimentally validated a universal 23S rRNA sequencing method, which included PCR amplification of the rDNA gene and direct cycle sequencing of the amplicon with eubacterial primers. The results of our sequence comparison indicated that the genus Listeria can be divided into two subgroups; one subgroup is composed of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri, whereas the other subgroup includes Listeria grayi subsp. grayi and Listeria grayi subsp. murrayi. A phylogenetic analysis revealed that these species diverged recently. These results are consistent with 16S rRNA sequence analysis data. For application purposes, one 16S rRNA region that can be sued to distinguish each Listeria species except L. Monocytogenes and L. innocua has been described. In this study we found four 23S rRNA signature regions which, when used in combination, can be used to distinguish the species.

Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains isolated in Brazil and France.

We evaluated the mutations in a 193bp of the rpoB gene by automated sequencing of rifampicin (RMP)-resistant and susceptible Mycobacterium tuberculosis strains isolated from Brazil (25 strains) and France (37 strains). In RMP-resistant strains, mutations were identified in 100% (16/16) from France and 89% (16/18) from Brazil. No mutation was detected in the 28 RMP-susceptible strains. Among RMP-resistant or RMP-susceptible strains deletion was observed. A double point mutation which had not been reported before was detected in one strain from France. Among French resistant strains mutations were found in codons 531 (31.2%), 526, 513 and 533 (18.7% each). In Brazilian strains the most common mutations were in codons 531 (72.2%), 526 (11.1%) and 513 (5.5%). The heterogeneity found in French strains may be related to the fact that most of those strains were from African or Asian patients.

Hydrophilic and cationic latex particles for the specific extraction of nucleic acids.

The adsorption of BSA and RNA onto hydrophilic and thermosensitive poly(N-isopropyl-acrylamide) (NIPAM) latex particles was described as a function of pH, ionic strength and temperature. The hydrogel poly(NIPAM) latex was synthesized by precipitation polymerization in the presence of a cationic amino-containing monomer. The latex obtained was characterized in terms of particle size, and electrophoretic mobility as a function of pertinent variables: pH, temperature and ionic strength. The adsorption of BSA onto the latex was investigated to identify the conditions at which the adsorbed amount of BSA was negligible. The adsorption of RNA was studied to establish the conditions which give rise to maximal adsorption of RNA. In order to favor the desorption of RNA, desorption was investigated by changing the pH, ionic strength, and temperature. The adsorption of BSA was found to be lower at 20 than at 40 degrees C. However, the adsorption of RNA is drastically affected by the pH and the ionic strength of the medium. Maximal adsorbed amounts were obtained at acidic pH, 20 degrees C, and low ionic strength. The adsorption is shown to decrease when the pH, temperature and ionic strength increase, implying that the adsorption was mainly governed by electrostatic interactions. Maximal release of RNA molecules was obtained at high ionic strength and basic pH.

Evidence of involvement of CD44 in endothelial cell proliferation, migration and angiogenesis in vitro.

Angiogenesis is essential for tumor growth and metastasis. In the process of angiogenesis, the interaction between adhesive proteins of endothelial cells and extracellular matrix components plays an important role by mediating cell attachment, which is indispensable for their motility, and by transmitting the regulatory signals for cell locomotion and proliferation. In this study, we examined the hypothesis that CD44 expressed on the endothelial cell surface is involved in the angiogenesis process. The experiments using calf pulmonary artery endothelial cells (CPAE) and a human microvascular endothelial cell line (HMEC-1) show that a monoclonal antibody against CD44 (clone J 173) inhibits endothelial cell proliferation by about 30% and migration by 25-50%, and abolishes the stimulating effect of hyaluronan polysaccharides on endothelial cell migration and proliferation. This antibody also suppresses the capillary formation of CPAE in an in vitro model of angiogenesis using fibrin matrix. These results provide evidence of the involvement of endothelial-cell-associated CD44 in angiogenesis.

Routine identification of Mycobacterium tuberculosis complex isolates by automated hybridization.

Methodologies for biochemical identification of mycobacteria isolated from clinical samples are still cumbersome, taking skilled technicians 3 to 6 weeks. We describe here a 2-h identification system for mycobacterial isolates belonging to the Mycobacterium tuberculosis complex using a DNA probe. After 30 min of hands-off sample preparation, the 1.5-h hybridization test is totally automated in the newly developed VIDAS system (bioMérieux, Marcyl'Etoile, France), which performs solid-phase specific hybridization of 16S rRNA at 37 degrees C. The strain collection of actinomycetes tested was composed of 662 isolates from 27 species: 461 members of the M. tuberculosis complex (443 M. tuberculosis, 10 M. bovis, and 8 M. bovis BCG isolates) and 201 isolates of other species, including 55 M. avium-intracellulare isolates). They were identified by traditional methods: growth rate, colonial morphology, pigmentation, and biochemical profiles. The automated probe assay displayed an excellent correlation with the reference results. The four members of the Nocardia and Rhodococcus genera tested did not cross-hybridize. This flexible random-access and automated technology was shown to suit the routine context of the laboratory by rapidly delivering the results.