Caracterización de plásmidos de acinetobacter baumannii en tres centros hospitalarios / Acinetobacter baumannii plasmids characterization in the hospitalary centers

Acinetobacter baumannii es un importante agente etiológico asociado a infecciones nosocomiales. Los patrones de resistencia a los principales agentes antimicrobianos y el perfil plasmídico fueron analizados en 34 cepas nosocomiales provenientes de tres hospitales del área metropolitana. La mayoría de los aislamientos (50 por ciento) provienen del tracto respiratorio y de la Unidad de Terapia Intensiva. Los porcentajes de resistencia obtenidos revelan la presencia de patrones complejos de resistencia múltiple incluso a los antimicrobianos imipenen, meropenen y ß lactamicos, los cuales son de uso frecuente en el tratamiento de las infecciones causadas por este germen. El perfil plasmídico obtenido sugiere una alta variabilidad. Un plásmido > 23 Kb fue asociado con la resistencia en algunos aislamientos. El análisis del DNA plasmídico es de gran utilidad en la tipificación epidemiológica de las cepas de A. baumannii y puede servir como un sistema complementario a los métodos epidemiológicos tradicionales

How bacteria protect themselves against channel-forming colicins.

Here we review the mechanisms that bacterial cells use to protect themselves against channel-forming colicins. Four mechanisms are examined: immunity, resistance, tolerance and PacB character. Immunity confers protection to colicinogenic cells against the colicin they produce, since the colicinogenic plasmid bears the genetic determinant for such immunity protein. Resistance is provided by modifications on colicin receptors located on the outer membrane. It prevents colicin adsorption and protects against those colicins sharing a common receptor. Tolerance is achieved by changes in the translocation system. The adsorbed colicin is not translocated toward the periplasmic space. This impedes its insertion into the cell membrane as well as the formation of the transmembrane channel. Tolerance confers protection against colicins that share the same translocation system. Finally, we discuss the PacB character, that confers protection against all known channel-forming colicins. The latter property is encoded by non-colicinogenic plasmids in the H-incompatibility complex.

On the mechanism of resistance to channel-forming colicins (PacB) and tellurite, encoded by plasmid Mip233 (IncHI3).

Plasmids of the H incompatibility complex confer protection against all known channel-forming colicins (PacB character) and resistance to potassium tellurite (Te(r)) to Escherichia coli strains. A DNA clone (2.2 kbp) from plasmid Mip233 (IncHI3) expressing PacB-Te(r) phenotypes was studied. DNA sequence analysis revealed a high degree of homology with the enzyme O-acetylserine sulfhydrylase. Size of the PacB-Te(r) transcript was estimated as 1200 bases. A single polypeptide was found on SDS-polyacrylamide gel with a molecular mass estimated of 34 kDa. The effect of channel-forming colicins and tellurite was analyzed at physiological and transcriptional levels. Results suggest that the pacB gene product could be a reductase-like enzyme. It is also suggested that presence of the PacB character among H plasmid confers selective advantage on cells sharing an ecological niche.

Construction of a cassette for cloning and analysis of replicons.

The aim of this work was the construction of a cassette, i.e., a non-replicative molecule formed by linkage of an antibiotic resistance gene and a multiple cloning site. This cassette would allow the cloning and analysis of a wide range of replicons. The aac(6')-lc amikacin gene was isolated and ligated to the multiple cloning site of the pUC18 vector. This construction was HindIII digested and cloned in the HindIII site of the vector. The resulting pHJ13 clone conferred to the recipient cells the ability to grow in presence of amikacin (cassette marker) and ampicillin (vector gene). By restriction analysis, the cassette orientation was established. Cassette versatility is provided by the presence of the unaltered multiple cloning site segment, and also because it allows sequencing of any replication origin inserted. Cassette functionality was demonstrated by ligation to a replicative region of H plasmid pHH1457. Presence of the ori region from pHH1457 and the aac(6')-lc gene was confirmed in E. coli transformed clones. The incompatibility properties of the pHH1457 and its capability to replicate in a Poll defective strain were preserved in the pHJII14 construct. Currently, the amikacin cassette is being used in the characterization of H Complex plasmids.

Cloning and characterization of a replicon region of the IncHII plasmid pHH1457.

A replicative region of the large conjugative plasmid pHH1457 (incompatibility group HII (IncHII)) was cloned. A 1.4-kbp region, in a stable pSBII14 clone, containing a PolI-independent replicon and determinants for the HII incompatibility phenotype, was selected and characterized. High incompatibility with IncHII plasmids was corroborated. Independent replication of the insert was demonstrated by ligation to an antibiotic resistance cassette. pSBII14 was used as a probe to identify IncHII plasmids from other members of the H complex: IncHI (IncHI1, IncHI2 and IncHI3 subgroups). Hybridization experiments revealed a high homology with the replication region of IncHII plasmids, but not with IncHI1 or IncHI3 plasmid prototypes. Homology with IncHI2 plasmids was observed, suggesting the presence of IncHII-like replicons among this subgroup of plasmids. This is the first report of the characterization of an IncHII plasmid maintenance region.

Cloning of the PacB-Ter region from plasmid Mip233 (IncHI3) and their expression in E. coli ton, tol mutants.

A region of the plasmid Mip233 (incompatibility group HI3) encoding the phenotypes of resistance to the channel-forming colicins (character PacB) and potassium tellurite (Ter), was cloned and studied. Both properties are contained in an insert of 2.2 Kbp, being the smallest functional clone (pB22) isolated so far. E. coli DH5 alpha pB22 transformants exhibit resistance to the colicins as well as to high levels of tellurite (> 1000 micrograms ml-1). Results suggest that they are genetically linked forming an inducible operon. pB22 does not show significant homology with DNA from other H plasmids. Tests using E. coli ton and tol mutants harbouring recombinant pB22 indicate that the product of gene tolC, but not that of tonB, is required for the expression of the PacB and Ter phenotypes.

Plasmid-encoded resistance to arsenic compounds in Gram-negative bacteria isolated from a hospital environment in Venezuela.

Resistance to arsenic compounds was examined among amikacin resistant Gram-negative bacteria isolate from a hospital environment. Arsenite resistance (Ars(r)) was found in a high proportion of isolates ( >60%) being frequently associated with resistance to tellurite (40%), and to other antimicrobial agents. Ars determinants (27%) were found to be transferable to E. coli K12 strains from which large plasmid DNA molecules were isolated and characterized by agarose gel electrophoresis. Plasmids were identified by both classical incompatibility tests, and by replicon typing using DNA specific probes. Most of the amikacin-arsenite (Ak-Ars) conjugative plasmids belong to the H incompatibility group. These results suggest that Ak-Ars resistance linked to IncH plasmids is wide spread in Gram-negative bacteria.

Detection of microcin production by pathogenic Escherichia coli isolates from children with acute diarrhoea.

Production of low molecular weight antibiotic substances was detected among pathogenic Escherichia coli isolates from diarrhoeal children's feces during evaluation of a rotavirus vaccine in Caracas city. One of these products, microcin V627a, was partially purified and characterized. Microcin synthesis and immunity system appears not to be plasmid determined and the antibiotic was produced to a high level in minimal medium during the stationary phase. This microcin has many features in common with the mccA15 family.

The incompatibility complex of H. plasmids.

This review deals with the general properties of the very large transfer thermosensitivity R. factor belonging to the H. incompatibility complex. This group is of particular interest not only because their temperature sensitivity transfer system but also for the number as well as distinctive resistance determinants being accumulate by them, and their prevalence in Salmonella serotypes and in other Gram-negative non-pathogenic bacteria both in man and animals.

PacB, a plasmid-encoded property which confers to E. coli K12 resistance to channel-forming colicins.

Plasmids belonging to incompatibility groups HI (subgroups HI2 and HI3 but not HI1) and HII but not from any other incompatibility group, confer to E. coli resistance against the bactericidal action of colicin B. Additionally it was demonstrated that this plasmid-encoded property, PacB, is not restricted to a specific colicin (colicin B) but also modifies bacterial susceptibility to the lethal activity determined by the channel-forming colicins A, B, E1, Ia, Ib, K, and N. PacB+ bacteria remain susceptible to colicins with other modes of action. This property appears to be highly conserved among the HI2 plasmids.

Characterization of polycyclic aromatic hydrocarbons degradative soil Pseudomonas.

Nine Pseudomonas strains, able to degrade polycycle aromatic hydrocarbons (PAHs), were isolated from enriched cultures with naphthalene, as carbon source, and soil samples from a land farming process applied on oil sludge, as inocula. Degradative tests showed that all the strains were capable to catabolize naphthalene (Nah) and phenanthrene (Phn). U2 strain transferred the selected function (Nah) to P. aeruginosa T1 (Hgr Oct+), however some of the transconjugants lost the Oct character, suggesting that it is of plasmidic nature. T1 derivatives as well the wild strains U28 and U31 transferred Nah function to P. putida AC165. All of the examined transconjugants also catabolized phenanthrene, suggesting that Nah and Phn functions in U2, U28, and U31 strains are linked and probably encoded by transferable plasmids.

pUCV11001, an IncH plasmid isolated in a Escherichia coli strain from a healthy child.

Plasmids conferring resistance to potassium tellurite but not to other antimicrobial agents were detected among E. coli multiresistant strains isolated from healthy children during a survey in Caracas. Few of them were autotransferable to E. coli K12 and they were conjugative only at temperatures below 30 degrees C. They also conferred to the host cells resistance to lethal action of colicin B, PacB character. pUCV11001, a prototype, was classified into the incompatibility group HI, subgroup HI2. Presence of these non-antimicrobial resistant IncH plasmids in E. coli from human sources is indicative of their wide distribution among Enterobacteria in nature.

Growth rate and production of a microcin by Salmonella typhimurium LT2 (strain M799-0) in continuous culture.

The production of a bacteriostatic microcin, of molecular weight 520 kD by Salmonella typhimurium LT2, strain M799-0, was studied in continuous culture. The effect of specific growth rate (mu) on microcin production was measured under aerobic and anaerobic conditions. In aerobiosis the microcin specific production rate (qp) was found to be strictly associated with cell growth. Thus, it is constitutively synthesized though at low production rate. As growth conditions become limited (i.e. anaerobiosis) its production is clearly induced and the highest yields are reached. Under the latter conditions microcin production follows a kinetic partial growth-linked process.