Frequency of mutation to rifampin resistance in Streptococcus pneumoniae clinical strains: hexA and hexB polymorphisms do not account for hypermutation.

The frequency of mutation to rifampin resistance of 200 clinical Streptococcus pneumoniae isolates was examined. Two peaks were observed in the distribution, with mode frequencies of 2.5 x 10(-7) (20% of isolates) and 2.5 x 10(-8). The hexA and hexB gene entire sequences were analyzed in 13 isolates. Sequences from both hypermutable and "normomutable" strains were conserved relative to that of the R6 S. pneumoniae control strain. The phenotypic Hex system proficiency, in terms of transforming efficiency, was also maintained irrespective of the variations in mutation frequency values.

Screening de infección tuberculosa en inmigrantes recién llegados a Ceuta / Screening of tuberculosis infection in recently arrived immigrants to Ceuta

Las poblaciones inmigrantes procedentes de países subsaharianos y del Magreb constituyen grupos de alto riesgo de infección tuberculosa. Analizamos la prevalencia de infección tuberculosa en un grupo de estos inmigrantes en Ceuta, con el fin de encontrar posibles diferencias en su comportamiento incidental según su país o región geográfica de procedencia y establecer protocolos y necesidad de estudio del estado de salud de dichos inmigrantes. Métodos: Estudio descriptivo transversal sobre una muestra de 2.223 inmigrantes (1.979 varones y 244 mujeres) procedentes de distintos países africanos (Nigeria, Mali, Guinea Bissau, Camerún, Ghana, Sierra Leona, Rep. Dem. Congo y Liberia, principalmente) en el campamento de refugiados de Calamocarro de Ceuta. Se practicó la intradermorreacción según la técnica de Mantoux según protocolo ya establecido, presentando los datos para tres puntos de corte.Resultados: Los inmigrantes procedían de 36 países africanos, el 89 por ciento eran varones y el 11 por ciento mujeres (p<0,001) con una edad media de 24,9 ñ 4,3 y 23,4 ñ 4,1 años, respectivamente. Un 32,6 por ciento presentaron una respuesta al test de Mantoux igual o superior a 10 mm, 33,2 por ciento en varones y 27,9 por ciento en mujeres (p=0,09). Sólo un 1,1 por ciento con induración 5-10 mm de probable origen vacunal. Por zonas geográficas, las tasas más altas se presentaron en inmigrantes procedentes de la R. D. Congo (65,1 por ciento) y Camerún (48,4 por ciento), seguidos a distancia por Nigeria (34, 0 por ciento), Liberia (32,7 por ciento), Mauritania (29,1 por ciento), Sierra Leona (28,8 por ciento), Costa de Marfil (27,8 por ciento), Guinea Bissau (27,4 por ciento), Ghana (26,3 por ciento), Argelia (25,6 por ciento), Mali (24,1 por ciento) y R. Guinea (20,9 por ciento) (p<0,0001). Conclusiones: La población inmigrante procedente de países centroafricanos presenta una alta prevalencia de infección tuberculosa, constituyendo un grupo de riesgo de padecer la enfermedad. Por tanto, es fundamental la implantación de programas específicos de búsqueda activa de infección tuberculosa durante su estancia en nuestra ciudad, aprovechando su concentración a la entrada en nuestro país, antes de su emigración definitiva a localidades penisulares y posterior dilución demográfica en la población de acogida. En dicho destino, el servicio médico supervisará el tratamiento a quién se le haya prescrito por el servicio del Área Funcional de Sanidad, eliminando así posibles futuras fuentes de infección de la enfermedad (AU)

[Prevalence of tuberculosis among the immigrant population in Ceuta, Spain]. / Prevalencia de infección tuberculosa en la población de inmigrantes en Ceuta, España.

BACKGROUND: The immigrant populations from sub-Saharan and Maghreb countries are groups in high risk of contracting tuberculosis. An analysis is made of the prevalence of tuberculosis infection among one group of these immigrants in Ceuta for the purpose of finding possible differences in their incidental behavior according to the country or geographical region from which they came. METHODS: A descriptive cross-sectional study of a sample of 2,223 immigrants (1979 males and 244 females) from different African countries (mainly Nigeria, Mali, Guinea-Bissau, Cameroon, Ghana, Sierra Leona, Democratic Republic of Congo and Liberia) at the Calamocarro refugee camp in Ceuta. A Mantoux intradermic reaction test was performed following the previously-established protocol, the data for three cut-off points being presented. RESULTS: The immigrants came from 36 African countries, 89.0% being males and 11.0% females (p < 0.001), respectively averaging in age from 24.9 +/- 4.3 to 23.4 +/- 4.1. A reaction to the Mantoux text of 10 mm or more was found in 32.6%, that is 33.2% among males and 27.9% among females (p = 0.09). Solely 1.1% showing 5-10 mm hardening of probable vaccination-related cause. By geographical regions, the highest rates were found among immigrants from the Democratic Republic of Congo (65.1%) and Cameroon (48.4%), followed far behind by Nigeria (34.0%), Liberia (32.7%), Mauritania (29.1%), Sierra Leona (28.8%), Ivory Coast (27.8%), Guinea-Bissau (27.4%), Ghana (26.3%), Algeria (25.6%), Mali (24.1%) and the Republic of Guinea (20.9%) (p < 0.0001). CONCLUSIONS: The immigrant population from central African countries shows a higher prevalence of tuberculosis infection, comprising a group at risk of contracting this disease. Therefore, it is of fundamental importance to implement specific programs to actively detect tuberculosis infection during their stay in our city, taking advantage of their being grouped together upon entry into our country, prior to their final emigration to localities throughout the mainland and subsequent mixing among the host country population.

A positive feedback mechanism controls expression of AlkS, the transcriptional regulator of the Pseudomonas oleovorans alkane degradation pathway.

The AlkS regulator, encoded by the alkS gene of the Pseudomonas oleovorans OCT plasmid, activates the expression of a set of enzymes that allow assimilation of alkanes. We show that the AlkS protein regulates, both negatively and positively, the expression of its own gene. In the absence of alkanes, alkS is expressed from promoter PalkS1, which is recognized by sigmaS-RNA polymerase, and whose activity is very low in the exponential phase of growth and considerably higher in stationary phase. AlkS was found to downregulate this promoter, limiting expression of alkS in stationary phase when alkanes were absent. In the presence of alkanes, AlkS repressed PalkS1 more strongly and simultaneously activated a second promoter for alkS, named PalkS2, located 38 bp downstream from PalkS1. Activation of PalkS2 allowed efficient transcription of alkS when alkanes were present. Transcription from PalkS2 was modulated by catabolite repression when cells were provided with a preferred carbon source. We propose that the expression of alkS is regulated by a positive feedback mechanism, which leads to a rapid increase in alkS transcription when alkanes are present. This mechanism should allow a rapid induction of the pathway, as well as a fast switch-off when alkanes are depleted. An improved model for the regulation of the pathway is proposed.

Resistance to tellurite as a selection marker for genetic manipulations of Pseudomonas strains.

Resistance to the toxic compound potassium tellurite (Telr) has been employed as a selection marker built into a set of transposon vectors and broad-host-range plasmids tailored for genetic manipulations of Pseudomonas strains potentially destined for environmental release. In this study, the activated Telr determinants encoded by the cryptic telAB genes of plasmid RK2 were produced, along with the associated kilA gene, as DNA cassettes compatible with cognate vectors. In one case, the Telr determinants were assembled between the I and O ends of a suicide delivery vector for mini-Tn5 transposons. In another case, the kilA and telAB genes were combined with a minimal replicon derived from a variant of Pseudomonas plasmid pPS10, which is able to replicate in a variety of gram-negative hosts and is endowed with a modular collection of cloning and expression assets. Either in the plasmid or in the transposon vector, the Telr marker was combined with a 12-kb DNA segment of plasmid pWW0 of Pseudomonas putida mt-2 encoding the upper TOL pathway enzymes. This allowed construction of antibiotic resistance-free but selectable P. putida strains with the ability to grow on toluene as the sole carbon source through an ortho-cleavage catabolic pathway.

Engineering of quasi-natural Pseudomonas putida strains for toluene metabolism through an ortho-cleavage degradation pathway.

To construct a bacterial catalyst for bioconversion of toluene and several alkyl and chloro- and nitro-substituted derivatives into the corresponding benzoates, the upper TOL operon of plasmid pWW0 of Pseudomonas putida was fully reassembled as a single gene cassette along with its cognate regulatory gene, xylR. The corresponding DNA segment was then targeted to the chromosome of a P. putida strain by using a genetic technique that allows deletion of all recombinant tags inherited from previous cloning steps and leaves the otherwise natural strain bearing exclusively the DNA segment encoding the phenotype of interest. The resulting strains grew on toluene as the only carbon source through a two-step process: conversion of toluene into benzoate, mediated by the upper TOL enzymes, and further metabolism of benzoate through the housekeeping ortho-ring cleavage pathway of the catechol intermediate.

Structure and function of the Pseudomonas putida integration host factor.

Integration host factor (IHF) is a DNA-binding and -bending protein that has been found in a number of gram-negative bacteria. Here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of IHF from Pseudomonas putida. Both the ihfA and ihfB genes of P. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, suggesting that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. Analysis of the amino acid differences between the E. coli and P. putida protein sequences suggests that in the evolution of IHF, amino acid changes were mainly restricted to the N-terminal domains and to the extreme C termini. These changes do not interfere with dimer formation or with DNA recognition. We constructed a P. putida mutant strain carrying an ihfA gene knockout and demonstrated that IHF is essential for the expression of the P(U) promoter of the xyl operon of the upper pathway of toluene degradation. It was further shown that the ihfA P. putida mutant strain carrying the TOL plasmid was defective in the degradation of the aromatic model compound benzyl alcohol, proving the unique role of IHF in xyl operon promoter regulation.

Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4.

The multimer resolution system (mrs) of the broad-host-range plasmid RP4 has been exploited to develop a general method that permits the precise excision of chromosomal segments in a variety of gram-negative bacteria. The procedure is based on the site-specific recombination between two directly repeated 140-bp resolution (res) sequences of RP4 effected by the plasmid-borne resolvase encoded by the parA gene. The efficiency and accuracy of the mrs system to delete portions of chromosomal DNA flanked by res sites was monitored with hybrid mini-Tn5 transposons in which various colored (beta-galactosidase and catechol 2,3 dioxygenase) or luminescent (Vibrio harveyi luciferase) phenotypic markers associated to res sequences were inserted in the chromosome of the target bacteria and exposed in vivo to the product of the parA gene. The high frequencies of marker excision obtained with different configurations of the parA expression system suggested that just a few molecules of the resolvase are required to achieve the site-specific recombination event. Transient expression of parA from a plasmid unable to replicate in the target bacterium was instrumental to effect differential deletions within complex hybrid transposons inserted in the chromosome of Pseudomonas putida. This strategy permits the stable inheritance of heterologous DNA segments virtually devoid of the sequences used initially to select their insertion.