The use of the replication region of plasmid pRS7 from Oenococcus oeni as a putative tool to generate cloning vectors for lactic acid bacteria.

A chimeric plasmid, pRS7Rep (6.1 kb), was constructed using the replication region of pRS7, a large plasmid from Oenococcus oeni, and pEM64, a plasmid derived from pIJ2925 and containing a gene for resistance to chloramphenicol. pRS7Rep is a shuttle vector that replicates in Escherichia coli using its pIJ2925 component and in lactic acid bacteria (LAB) using the replication region of pRS7. High levels of transformants per µg of DNA were obtained by electroporation of pRS7Rep into Pediococcus acidilactici (1.5 × 10(7)), Lactobacillus plantarum (5.7 × 10(5)), Lactobacillus casei (2.3 × 10(5)), Leuconostoc citreum (2.7 × 10(5)), and Enterococcus faecalis (2.4 × 10(5)). A preliminary optimisation of the technical conditions of electrotransformation showed that P. acidilactici and L. plantarum are better transformed at a later exponential phase of growth, whereas L. casei requires the early exponential phase for better electrotransformation efficiency. pRS7Rep contains single restriction sites useful for cloning purposes, BamHI, XbaI, SalI, HincII, SphI and PstI, and was maintained at an acceptable rate (>50%) over 100 generations without selective pressure in L. plantarum, but was less stable in L. casei and P. acidilactici. The ability of pRS7Rep to accept and express other genes was assessed. To the best of our knowledge, this is the first time that the replication region of a plasmid from O. oeni has been used to generate a cloning vector.

Optimization of technical conditions for the transformation of Pediococcus acidilactici P60 by electroporation.

Previously reported techniques for the electrotransfer of foreign DNA into pediococci yield only a small number of transformants/mug DNA, especially when using undomesticated strains. This study reports an improved protocol for the electrotransformation of pediococci, based on trials using Pediococcus acidilactici P60 and the plasmid pRS4C1. The improved protocol yields from 2 to 3 log units more transformants than the previously reported methods, with up to (9.1+/-1.3)x10(4) transformants/mug of foreign DNA under the best conditions identified. The most important modifications proposed are an increase in electric field strength during electroporation (from 12.5 to 20kV/cm) and a reduction in lysozyme concentration during the preparation of electrocompetent cells (from 4000 to 2000U/ml): together, these two modifications greatly improve transformant yield. In addition, increasing cell culture time (from OD(600nm)=0.6 to OD(600nm)=1.0-1.2) and increasing dl-threonine concentration in the growth medium (from 20 to 40mM) also contribute to improved electrotransformation efficiency.

Nucleotide sequence, structural organization and host range of pRS4, a small cryptic Pediococcus pentosaceus plasmid that contains two cassettes commonly found in other lactic acid bacteria.

The complete nucleotide sequence of the cryptic plasmid pRS4 (3550 bp) from Pediococcus pentosaceus RS4 was determined. Sequence analysis revealed the presence of three open reading frames (ORFs). The putative protein coded by ORF 1 showed 93% identity with the mobilization protein of Lactobacillus casei plasmid pLC88 and 94% identity with a sequenced fragment of the mobilization protein of P. damnosus plasmid pF8801, suggesting a common origin for these three mobilization proteins. The putative protein coded by ORF 2 showed 92% identity with the replication protein of L. plantarum plasmid pWCFS101, a plasmid that replicates via the rolling circle (RC) mechanism, suggesting a similar replication mechanism for pRS4. Supporting this hypothesis, a putative double strand origin (dso) and a region with palindromic sequences that could function as single strand origin (sso), were detected in pRS4. A function could not be assigned to ORF 3. Since ORF 1 exhibits high identity with L. casei plasmid pLC88 but lower identity (58%) with other Lactobacillus plasmids, and ORF 2 exhibits high identity with the L. plantarum plasmid pWCFS101 but lower identity (55-58%) with other Lactobacillus plasmids (including pLC88), two independent cassettes, from different sources, seem to be involved in the structure of pRS4. Plasmids derived from pRS4 containing the chloramphenicol resistance gene were successfully electrotransformed in L. plantarum, L. casei, P. pentosaceus, and Pediococcus acidilactici, suggesting that pRS4 could be used as a cloning vector for lactic acid bacteria. To our knowledge pRS4 is the first RC-replicating plasmid of P. pentosaceus that has been completely sequenced and used as cloning vector.

Transformation of Lactobacillus plantarum by electroporation with in vitro modified plasmid DNA.

An improved method for the electrotransformation of Lactobacillus plantarum CECT 220 (ATCC 8014) with plasmid DNA isolated from Escherichia coli is described. The two main modifications with respect to existing methods are: (i) isolation of plasmid DNA from E. coli JM110 grown in minimal medium and (ii) in vitro modification of the DNA by cell-free extracts of the host L. plantarum. Optimal electrotransformation was obtained with exponentially growing cells of L. plantarum concentrated to 6x10(9) cfu ml-1, with electric pulses of 13 kV cm-1 in cuvettes with 1 mm inter-electrode distance. We consider that this method constitutes a useful tool for routine manipulation of L. plantarum, and can probably be extended to other lactic acid bacteria.

[Efficacy preemptive therapy with ganciclovir for the prevention of cytomegalovirus disease in liver transplant recipients]. / Eficacia del tratamiento anticipado con ganciclovir en la prevención de la enfermedad por citomegalovirus en receptores de un trasplante de hígado.

BACKGROUND AND OBJECTIVE: In liver transplant recipients the most frequent infection is that produced by cytomegalovirus (CMV). One of the methods to reduce the incidence of that infection is the CMV pp65 antigenemia-guided preemptive therapy with intravenous ganciclovir. PATIENTS AND METHOD: Liver transplant recipients were tested for CMV antigenemia at days 14, 28, 45, 60, 90 and 180 postransplantation and when clinically indicated. Patients showing > 50/200.000 leukocytes received ganciclovir 5 mg/kg/12 h for 14 days. Risk factors for active CMV disease where studied. RESULTS: 182 CMV seropositive patients where included in the study. 16 patients with > 50/200.000 leukocytes received ganciclovir as preemptive therapy. CMV disease appeared in 9/182 patients (4.9%): 2/16 who received PT and 7/166 among those who did not receive preemptive therapy. The only factor associated with increased incidence of CMV disease was to have missing samples for CMV antigenemia during the follow up (p < 0.0042; OR = 8,17; 95% CI, 1.94-34.36). CONCLUSIONS: Ganciclovir antigenemia-guided preemptive therapy is associated with a low incidence of CMV disease. Bad adherence to the protocol of antigenemia samples increases the risk for CMV disease.

Plasmid curing of Oenococcus oeni.

Two strains of Oenococcus oeni, RS1 (which carries the plasmid pRS1) and RS2 (which carries the plasmids pRS2 and pRS3), were grown in the presence of different curing agents and at different temperatures. Sublethal temperature together with acriflavine generated all possible types of cured strains, i.e., lacking pRS1 (from strain RS1), and lacking pRS2, pRS3, or both (from strain RS2). Sublethal temperature together with acridine orange only generated cured strains lacking pRS3. These results suggest that acriflavine is a better curing agent than acridine orange for O. oeni, and that pRS3 is the most sensitive to these curing agents. We also observed spontaneous loss of pRS2 or both pRS2 and pRS3 by electroporation. The ability to cure O. oeni strains of plasmids provides a critical new tool for the genetic analysis and engineering of this commercially important bacterium.

Eficacia del tratamiento anticipado con ganciclovir en la prevención de la enfermedad por citomegalovirus en receptores de un trasplante de hígado / Efficacy preemptive therapy with ganciclovir for the prevention of cytomegalovirus disease in liver transplant recipients

FUNDAMENTO Y OBJETIVO: La infección por citomegalovirus (CMV) es la complicación infecciosa más frecuente en los receptores de un trasplante hepático. Para disminuir la incidencia de esta infección uno de los métodos es el tratamiento anticipado con ganciclovir guiado por la determinación seriada de antigenemia pp65 de CMV. PACIENTES Y MÉTODO: En los receptores de un trasplante hepático seropositivos para CMV antes del trasplante, se realizaron determinaciones de antigenemia pp65 de CMV los días 14, 28, 45, 60, 90 y 180 postrasplante y cuando se consideró indicado clínicamente. Se realizó tratamiento anticipado con ganciclovir intravenoso (5 mg/kg de peso cada 12 h durante 14 días) en todos los pacientes que tuvieron antigenemia superior a 50 células/200.000 leucocitos. Se analizaron los factores de riesgo para el desarrollo de enfermedad activa por CMV. RESULTADOS: Se incluyó en el estudio a 182 pacientes seropositivos para CMV. Se realizó tratamiento anticipado con ganciclovir en 16 pacientes con antigenemia superior a 50 células/200.000 leucocitos. Presentaron enfermedad activa por CMV 9 de 182 pacientes (4,9 por ciento): 2 de los 16 que recibieron tratamiento anticipado con ganciclovir y 7 de los 166 que no lo recibieron. El único factor de riesgo asociado de manera significativa con enfermedad activa por CMV fue la no realización de alguna de las antigenemias previstas (p < 0,0042; odds ratio = 8,17; intervalo de confianza del 95 por ciento 1,94-34,36). CONCLUSIONES: El tratamiento anticipado con ganciclovir se asocia a una baja incidencia de enfermedad por CMV. La mala adherencia al protocolo de realización seriada de antigenemia pp65 aumenta el riesgo de enfermedad por CMV (AU)