[Characterization of plasmids which mediate resistance to multiple antibiotics in gram-negative bacteria of nosocomial origin]. / Caracterización de plásmidos que median la resistencia a múltiples antibióticos en bacterias gramnegativas de origen nosocomial.

BACKGROUND: The genetic and molecular mechanisms involved in antimicrobial resistance of 10 strains of gramnegative bacilli (1 Serratia marcescens; 2 Escherichia coli; 1 Proteus mirabilis; 4 Klebsiella pneumoniae; 1 Enterobacter cloacae y 1 Alcaligenes faecalis), isolated from adult patients with nosocomial pulmonary infection at the in-patient facilities of the University Hospital of Los Andes, Mérida, Venezuela, have been studied. METHODS: The antimicrobial susceptibility was determined by minimum inhibitory concentrations using the dilution method in agar. The study of extrachromosomal genes was carried out by conjugation, bacterial infection with the bacteriophage M13 and curing of plasmid by acridine orange. The plasmids were isolated by alkaline lysis and analysis of restriction endonuclease digestion was carried out separately using the enzymes EcoRI and HindIII. A DNA probe, derived from the region which encodes the TEM-1 beta-lactamase of the plasmid pBR322 was used for dot-blot hybridization tests. RESULTS: All of the gramnegative bacilli showed resistance to ampicillin, carbenicillin and cephalothin (> 128 micrograms/ml) and 3 strains also showed resistance to gentamicin (> 64 micrograms/ml). Genetic and molecular procedures showed the presence of conjugative plasmids of approximately 54 kb in all the 10 strains. The restriction patterns obtained by using EcoRI and HindIII indicated common DNA fragments in most of the plasmids studied. The dot-blot hybridization tests confirmed homology between the plasmids and the DNA probe used (TEM-1 beta-lactamase). CONCLUSIONS: In this study, the gramnegative bacteria of nosocomial origin harbored self-transferable plasmids of approximately 54 kb, which mediate resistance to gentamicin and encode a beta-lactamase of the TEM group.

[In vivo cloning of genes of Escherichia coli K12 by means of homologue recombination]. / Cloneo in vivo de genes de Escherichia coli K12 mediante recombinación homóloga.

Plasmid pT153, a stable recombinant between pBR322 plasmid and M13 bacteriophage, and Tn10 transposon were employed for in vivo cloning of a chromosomal segment of Escherichia coli including the nar locus. The strategy consisted in creating an homology between pT153 and the E. coli chromosome, incorporating a Tn10 transposon close to the nar locus of a polA12 temperature-sensitive strain. The selection of clones carrying the plasmid into the chromosome was realized at 42 degrees C, with ampiciline, taken advantage that the replicon of pT153 requires the ADN Polymerase I for its functioning. The plasmid integrated in the polA12 strain has the opportunity of excising and carrying part of bacterial genome and autonomically replicating after a thermal change from 42 degrees C to 30 degrees C. The stable recombinant plasmids could be efficiently transduced with the M13 bacteriophage to a E. coli strain (delta narGHJI), obtaining Nit+ Apr transductant clones. The versatility of this method of E. coli gene cloning is the facility to create the homologue region anywhere in the bacterial genome and the efficient transduction of pT153 plasmid by M13 bacteriophage.

[Obtention of Trypanosoma cruzi clones of thin and thick epimastigotes and trypomastigotes living in the blood on various agar media]. / Obtención de clones de Trypanosoma cruzi a partir de epimastigotes y tripomastigotes sanguícolas delgados y gruesos en diferentes medios agarizados.

Trypanosoma cruzi (EP strain) presents clonal growth when it is incorporated into at 0.7% agar and at 0.8% in surface. The colonies were obtained from a single parasite and in a relatively short time. The efficiency of plating was variable and depended on the type of agar medium. A maximal efficiency was obtained for at 0.7 agar/LIT-BHI and for at 0.8% agar/LIT-BHI-Blood. The sanguicolous tripomastigotes, either thin or thick, were able to generate colonies from single parasites, with 100% plating efficiency, higher than 80% corresponding to the epimastigotes coming from LIT medium.

[Stable recombinants of bacteriophage M13 and plasmid pBR322]. / Recombinantes estables entre el bacteriofago M13 y el plásmido pBR322.

Two recombinants between the phage M13 and the plasmid pBR322 were isolated, analyzing the plasmid content of over one hundred colonies obtained by transduction. The study of the structure of both recombinants indicates that a fragment of the M13 genome has been integrated to pBR322. In both cases, the fragment contains a part of the phage replication region inserted either in the vicinity or within the pBR322 replicon. The fact that the phage and plasmid replicons seem to be involved in the recombination event suggests that it is helpful when the replication begins. So far it has not been possible to isolate a recombinant taking the whole genomes of pBR322 and M13. This is, undoubtedly, due to the instability of the recombinant molecule.

Replication functions of pC194 are necessary for efficient plasmid transduction by M13 phage.

Escherichia coli plasmids pBR313 and pBR322 were transduced by phage M13 with low efficiency (10(-8) transductants/phage). Hybrid plasmids pHV12 or pHV33, composed of Staphylococcus aureus plasmid pC194 and pBR313 or pBR322, respectively, were transduced much more efficiently (10(-4) transductants/phage). Inactivation of either of the two zones necessary for pC194 replication, one coding for a protein, the other not, reduced the transforming efficiency of hybrids to the level of pBR322. Activity of the pC194 replication region was not necessary for the formation of chimeras between M13 and the transduced plasmid in the donor cells, but rather for the establishment of the plasmid in the recipient cells.

Genes involved in transitory recombination between phage M13 and plasmid pHV33.

Plasmid pHV33 and phage M13 combine in Escherichia coli cells to form a chimera, which decombines to regenerate two parental genomes. Combination can occur via two genetic pathways, one defined by the recBC genes, the other by recA, recF and possibly recL genes. Decombination can also occur via two pathways, one defined again by the recBC genes, the other by a gene not identified, but active only in the absence of the recL gene product.

Transitory recombination between plasmid pHV33 and phage M13.

Plasmid pHV33 and phage M13 which have no homology exceeding 13 bp, combine in Escherichia coli cells. The chimeric genome is encapsidated in phage proteins and injected into a recipient cell, where it decombines to regenerate the two parental genomes. We call this combination-decombination process 'transitory recombination'.

Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells.

Escherichia coli cells are 4--6 times more transformable and 20--30 times more competent after 24 h incubation in cold calcium chloride than immediately after calcium chloride treatment. With 24-h-old competent cells we obtained routinely 2 . 10(7) transformants per microgram of pBR322 DNA, and transformed over 20% of viable cells.