Draft Genome Sequences of Two Epidemic OXA-48-Producing Klebsiella pneumoniae Clinical Strains Isolated during a Large Outbreak in Spain.

We report here the draft genome sequences of Klebsiella pneumoniae strains Kp1803 and Kp3380 isolated during a large outbreak at A Coruña Hospital in Spain. The final genome assemblies for Kp1803 and Kp3380 comprise approximately 6.6 and 6.1 Mb, respectively, and both strains have G+C contents of 57.2%.

The FhaB/FhaC two-partner secretion system is involved in adhesion of Acinetobacter baumannii AbH12O-A2 strain.

Acinetobacter baumannii is a hospital-acquired pathogen that shows an extraordinary capacity to stay in the hospital environment. Adherence of the bacteria to eukaryotic cells or to abiotic surfaces is the first step for establishing an infection. The A. baumannii strain AbH12O-A2 showed an exceptional ability to adhere to A549 epithelial cells. The AbFhaB/FhaC 2-partner secretion (TPS) system involved in adhesion was discovered after the screening of the recently determined A. baumannii AbH12O-A2 strain genome (CP009534.1). The AbFhaB is a large exoprotein which transport to the bacterial surface is mediated by the AbFhaC protein. In the present study, the role of this TPS system in the AbH12O-A2 adherence phenotype was investigated. The functional inactivation of this 2-partner secretion system was addressed by analyzing the outer membrane vesicles (OMV) proteomic profile from the wild-type strain and its derivative mutant AbH12O-A2ΔfhaC demonstrating that AbFhaB is no longer detected in the absence of AbFhaC. Scanning electron microscopy (SEM) and adhesion experiments demonstrated that inactivation of the AbFhaB/FhaC system significantly decreases bacterial attachment to A549 alveolar epithelial cells. Moreover, it has been demonstrated that this 2-partner secretion system is involved in fibronectin-mediated adherence of the A. baumannii AbH12O-A2 isolate. Finally, we report that the AbFhaB/FhaC system is involved in virulence when tested using invertebrate and vertebrate hosts. These data suggest the potential role that this AbFhaB/FhaC secretion system could play in the pathobiology of A. baumannii.

Genome Sequence of a Clinical Strain of Acinetobacter baumannii Belonging to the ST79/PFGE-HUI-1 Clone Lacking the AdeABC (Resistance-Nodulation-Cell Division-Type) Efflux Pump.

Increased expression of chromosomal genes for resistance-nodulation-cell division-type efflux systems plays a major role in the multidrug resistance of Acinetobacter baumannii Little is known about the genetic characteristics of clinical strains of Acinetobacter baumannii lacking the AdeABC pump. In this study, we sequenced the genome of clinical strain Ab421 GEIH-2010 (belonging to clone ST79/PFGE-HUI-1 from the GEIH-REIPI Ab. 2010 project) which lacks this efflux pump.

Complete Genome Sequence of the Multiresistant Acinetobacter baumannii Strain AbH12O-A2, Isolated during a Large Outbreak in Spain.

We report the complete genome sequence of Acinetobacter baumannii strain AbH12O-A2, isolated during a large outbreak in Spain. The genome has 3,875,775 bp and 3,526 coding sequences, with 39.4% G+C content. The availability of this genome will facilitate the study of the pathogenicity of the Acinetobacter species.

Attitudes to rapid HIV testing among Spanish General Practitioners.

OBJECTIVES: The objectives of this study were to investigate the acceptability of rapid HIV testing among general practitioners in Spain and to identify perceived barriers and needs in order to implement rapid testing in primary care settings. METHODS: An anonymous questionnaire was distributed online to all members of the two largest Spanish scientific medical societies for family and community medicine. The study took place between 15th June and 31st October 2010. RESULTS: Completed questionnaires were returned by 1308 participants. The majority (90.8%) of respondents were General Practitioners (GP). Among all respondents, 70.4% were aware of the existence of rapid tests for the diagnosis of HIV but they did not know how to use them. Nearly 80% of participants would be willing to offer rapid HIV testing in their practices and 74.7% would be confident of the results obtained by these tests. The barriers most commonly identified by respondents were a lack of time and a need for training, both in the use of rapid tests (44.3% and 56.4%, respectively) and required pre- and post-test counselling (59.2% and 34.5%, respectively). CONCLUSIONS: This study reveals a high level of acceptance and willingness on the part of GPs to offer rapid HIV testing in their practices. Nevertheless, the implementation of rapid HIV testing in primary care will not be possible without moving from comprehensive pre-test counselling towards brief pre-test information and improving training in the use of rapid tests.

Acceptability of rapid HIV diagnosis technology among primary healthcare practitioners in Spain.

This study investigated the acceptability of rapid HIV testing among general practitioners (GP) and aimed to identify perceived barriers and needs in order to implement rapid testing in primary care settings. An anonymous questionnaire was distributed online to all members of the two largest Spanish scientific medical societies for family and community medicine. The study took place between 15 June 2012 and 31 October 2010. Completed questionnaires were returned by 1308 participants. The majority (90.8%) of respondents were GP. Among all respondents, 70.4% were aware of the existence of rapid tests for the diagnosis of HIV but they did not know how to use them. Nearly 80% of participants would be willing to offer rapid HIV testing in their practices and 74.7% would be confident of the result obtained by these tests. The barriers most commonly identified by respondents were a lack of time and a need for training, both in the use of rapid tests (44.3% and 56.4%, respectively) and required pre- and post-test counselling (59.2% and 34.5%, respectively). This study reveals a high level of acceptance and willingness on the part of GPs to offer rapid HIV testing in their practices. Nevertheless, the implementation of rapid HIV testing in primary care will not be possible without moving from comprehensive pre-test counselling towards brief pre-test information and improving training in the use of rapid tests.

Susceptibility to the Cry1F toxin of field populations of Sesamia nonagrioides (Lepidoptera: Noctuidae) in Mediterranean maize cultivation regions.

Maize hybrids expressing the Cry1F toxin provide efficient control of lepidopteran pests. The Mediterranean corn borer, Sesamia nonagrioides (Lefèvre), is one of the most damaging pests of maize in the Mediterranean basin. In this work we firstly determined the efficacy of maize hybrids expressing the Cry1F toxin (event TC1507) to control neonates of S. nonagrioides. Leaf tissue feeding bioassays revealed that TC1507 maize is highly effective against this pest, and the percentage mortality obtained was comparable to that obtained with a Cry1Ab-expressing maize hybrid (Compa CB, event 176), which is known to be highly efficacious against S. nonagrioides. Secondly, interpopulation variation in the susceptibility to the Cry1F insecticidal protein was established for nine field-collected populations of S. nonagrioides (three Spanish, two French, two Italian, one Greek, and one Turkish). Estimates of the susceptibility of larvae to the Cry1F toxin showed low variability in lethal concentrations and growth inhibition concentrations among field populations. Moreover, no significant differences were found when they were grouped by geographical areas [Western Mediterranean (Spain and France) versus Eastern Mediterranean (Italy, Greece and Turkey)] or by history of exposure to Bt plants (Spanish vs. other populations). Therefore, the minor differences found in field populations can be attributed to natural variation in sensitivity to Cry1F. The importance of establishing baselines of susceptibility for resistance detection is discussed. Future changes in susceptibility of S. nonagrioides populations to Cry1F could be documented based on this baseline data.

Short communication: A comparative analysis of recombinant chymosins.

The first step in cheesemaking is the milk clotting process, in which κ-caseinolytic enzymes contribute to micelle precipitation. The best enzyme for this purpose is chymosin because of its high degree of specificity toward κ-casein. Although recombinant bovine chymosin is the most frequently used chymosin in the industry, new sources of recombinant chymosin, such as goat, camel, or buffalo, are now available. The present work represents a comparative study of 4 different recombinant chymosins (goat and buffalo chymosins expressed in Pichia pastoris, and bovine and camel chymosin expressed in Aspergillus niger). Recombinant goat chymosin exhibited the best catalytic efficiency compared with the buffalo, bovine, or camel recombinant enzymes. Moreover, recombinant goat chymosin exhibited the best specific proteolytic activity, a wider pH range of action, and a lower glycosylation degree than the other 3 enzymes. In conclusion, we propose that recombinant goat chymosin represents a serious alternative to recombinant bovine chymosin for use in the cheesemaking industry.

[Postsurgical pituitary apoplexy. Report of two cases]. / Apoplejía tumoral hipofisaria posquirúrgica. A propósito de dos casos.

We report two cases of large macroadenomas that, after a transsphenoidal partial resection, suffered necrosis and swelling of the residual tumor, with increase of its volume, compression of neighboring structures and neurological deterioration. The literature is reviewed looking for possible pathophysiological mechanism and prevention.

Apoplejía tumoral hipofisaria posquirúrgica. A propósito de dos casos / Postsurgical pituitary apoplexy. Report of two cases

Se presentan dos casos de macroadenomas nofuncionantes grandes que, tras su extirpación parcialpor vía transesfenoidal, sufrieron un infarto delresto tumoral residual, con resultado de aumento desu volumen, compresión de las estructuras vecinas ydeterioro neurológico de los pacientes. Ambos fueronreintervenidos por vía transcraneal para conseguirla extirpación completa de sus tumores. Se revisa labibliografía en busca de las claves para prevenir estacomplicación (AU)

We report two cases of large macroadenomasthat, after a transsphenoidal partial resection, sufferednecrosis and swelling of the residual tumor, withincrease of its volume, compression of neighboringstructures and neurological deterioration. The literatureis reviewed looking for possible pathophysiologicalmechanism and prevention (AU)

[Pituitary adenoma apoplexy after spinal anaesthesia. Report of two cases and review of the literature]. / Apoplejía de adenoma hipofi sario no funcionante tras anestesia espinal. A propósito de dos casos.

Two cases of pituitary apoplexy occurring after spinal anaesthesia are described. Both presented with headache and III par palsy, one of them with additional visual disturbance and hyponatremia. A bibliographical review has been done, looking for early diagnostic keys. We propose the arterial hypotension as a possible pathogenetic mechanism of pituitary adenoma apoplexy.

Apoplejía de adenoma hipofi sario no funcionante tras anestesia espinal. A propósito de dos casos / Pituitary adenoma apoplexy after spinal anaesthesia. Report of two cases and review of the literature

Se presentan dos casos clínicos de apoplejía hipofisaria tras anestesia espinal, ambos con cefalea y unaparálisis del III par craneal. Uno de ellos con alteraciónvisual adicional e hiponatremia. Se revisa la bibliografíaen busca de las claves para un diagnóstico precozy se propone la hipotensión arterial como mecanismopatogénico de la apoplejía (AU)

Two cases of pituitary apoplexy occurring afterspinal anaesthesia are described. Both presented withheadache and III par palsy, one of them with additionalvisual disturbance and hyponatremia. A bibliographicalreview has been done, looking for early diagnostickeys. We propose the arterial hypotension as a possiblepathogenetic mechanism of pituitary adenoma apoplexy (AU)

Purification and characterization of a milk-clotting protease from Bacillus licheniformis strain USC13.

AIM: The study of a milk-clotting protease secreted by Bacillus licheniformis strain USC13. METHODS AND RESULTS: Growth of B. licheniformis USC13 in LB medium resulted in the production of a serine protease with a molecular weight of 62 kDa processed to its mature form of 34 kDa, both forms were found in the extracellular medium. The enzyme exhibited typical milk-clotting kinetics. CONCLUSIONS: The capacity of this protease to produce milk curds could make it useful as a new source of milk coagulants. SIGNIFICANCE AND IMPACT OF THE STUDY: Cheese-making industry seeks for novel enzyme sources, and microbial coagulants have several advantages over animal and plant counterparts. The protease from B. licheniformis has the ability to produce milk curds although more studies about quality of both the enzyme and the milk curds formed should be carried out in the future to confirm its usefulness in the dairy industry.

Cloning and expression of the XPR2 gene from Yarrowia lipolytica in Pichia pastoris.

Yarrowia lipolytica is a dimorphic yeast able to secrete different types of proteases depending on the pH of the environment. At neutral pH, the production of an extracellular alkaline protease (AEP) is induced. This protease could be useful in the leather, detergent, or food industries. The XPR2 gene, coding for AEP, was extracted from the pINA154 vector and cloned into the pHIL-D2 vector to obtain a new protease-producing recombinant Pichia pastoris strain. The gene was efficiently integrated in the P. pastoris genome and expressed from the AOX1 promoter actively induced by methanol. Finally, the protease was successfully secreted by P. pastoris GS115.

Fluorescein thiocarbamoyl-kappa-casein assay for the specific testing of milk-clotting proteases.

Milk-clotting proteases, which are widely used in the cheese-making industry, are enzymes that use soluble caseins as their preferential substrates. Here, we propose a modification to a method previously described for the specific determination of milk-clotting proteases by using kappa-casein labeled with fluorescein isothiocyanate as substrate. Validation of the modified method was confirmed using natural bacterial, fungal, plant, and animal milk-clotting proteases, as well as a milk-clotting enzyme of recombinant origin. The new modified method described here allowed specific quantification of the activity of milk-clotting proteases in a very sensitive way and permitted determination of the appropriate kinetic parameters of all the enzymes tested, consistent with their origin and degree of purity.

Proposal of a method for the genetic transformation of Gordonia jacobaea.

AIMS: Gordonia jacobaea is a recently isolated bacterial species with potential industrial application on account of its ability to store large quantities of trans-canthaxanthin. Its genetic manipulation is, however, difficult and cumbersome owing to the presence of mycolic acids in the cell wall and, especially, because of current lack of knowledge about its basic genetics. The present work describes a method for the genetic transformation of G. jacobaea. METHODS AND RESULTS: Gordonia jacobaea was grown in media supplemented with different glycine, penicillin G and isoniazid concentrations. The temperature, carbon source, growth phase and ultrasounds were analyzed for improving the method efficiency. The cells were finally transformed by electroporation. Finally, the method was applied to Brevibacteriumlactofermentum and Gordonia bronchialis. CONCLUSIONS: The growth of G. jacobaea in the presence of glycine and isoniazid is essential for obtaining electrocompetents cells. The temperature, growth phase and ultrasounds appeared as the main factors for increasing the transformation efficiency. The use of shuttle plasmids became necessary. The method described can be used with other Corynebacteria species. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the importance of the CNM group (Corynebacteria, Nocardia and Mycobacteria genera) in different areas such as industry, bioremediation improve the knowledge of their molecular mechanisms are becoming essential. The method described here improves the genetic manipulation of this group of bacteria.

Meningiomas after radiation-therapy for benign astrocytomas.

A 4.5 year-old-girl was submitted to subtotal removal of a benign astrocytoma of the left temporal lobe with basal ganglia extension and given radiotherapy. At age 27, she was found to harbor a right posterior frontal meningioma that was totally removed. A recurrence of the right convexity meningioma was again excised after a 4-year interval. The authors report this case to illustrate the possibility of the appearance of radiation-induced meningiomas after an interval of 22 years and briefly discuss 16 previous reports on this occurrence in benign astrocytomas.

Diversity of trypsins in the Mediterranean corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae), revealed by nucleic acid sequences and enzyme purification.

The existence of a diverse trypsin gene family with a main role in the proteolytic digestion process has been proved in vertebrate and invertebrate organisms. In lepidopteran insects, a diversity of trypsin-like genes expressed in midgut has also been identified. Genomic DNA and cDNA trypsin-like sequences expressed in the Mediterranean corn Borer (MCB), Sesamia nonagrioides, midgut are reported in this paper. A phylogenetic analysis revealed that at least three types of trypsin-like enzymes putatively involved in digestion are conserved in MCB and other lepidopteran species. As expected, a diversity of sequences has been found, including four type-I (two subtypes), four type-II (two subtypes) and one type-III. In parallel, four different trypsins have been purified from midgut lumen of late instar MCB larvae. N-terminal sequencing and mass spectrometric analyses of purified trypsins have been performed in order to identify cDNAs coding for major trypsins among the diversity of trypsin-like sequences obtained. Thus, it is revealed that the four purified trypsins in MCB belong to the three well-defined phylogenetic groups of trypsin-like sequences detected in Lepidoptera. Major active trypsins present in late instar MCB lumen guts are trypsin-I (type-I), trypsin-IIA and trypsin-IIB (type-II), and trypsin-III (type-III). Trypsin-I, trypsin-IIA and trypsin-III showed preference for Arg over Lys, but responded differently to proteinaceous or synthetic inhibitors. As full-length cDNA clones coding for the purified trypsins were available, three-dimensional protein models were built in order to study the implication of specific residues on their response to inhibitors. Thus, it is predicted that Arg73, conserved in type-I lepidopteran trypsins, may favour reversible inhibition by the E-64. Indeed, the substitution of Val213Cys, unique for type-II lepidopteran trypsins, may be responsible for their specific inhibition by HgCl2. The implication of these results on the optimisation of the use of protease inhibitors for pest control, and on the identification of endoprotease-mediated resistance to Bacillus thuringiensis Cry-toxins is discussed.

Meningiomas after radiation-therapy for benign astrocytomas / Meningiomas tras radioterapia en astrocitomas benignos

Se presenta el caso de una niña de 4 años y medio que recibió radioterapia local tras la resección casi total de un astrocitoma benigno temporal izquierdo con extensión a ganglios basales. A los 27 años fue operada con extirpación macroscópica total de un meningioma frontal posterior derecho, que recidivó y fue reoperado 4 años más tarde. El propósito de la comunicación es comentar la posibilidad de la aparición de un meningioma radioinducido 22 años después de la radioterapia y revisar brevemente 16 casos similares de meningioma tras la radioterapia en astrocitomas benignos

A 4.5 year-old-girl was submitted to subtotal removal of a benign astrocytoma of the left temporal lobe with basal ganglia extension and given radiotherapy. At age 27, she was found to harbor a right posterior frontal meningioma that was totally removed. A recurrence of the right convexity meningioma was again excised after a 4-year interval. The authors report this case to illustrate the possibility of the appearance of radiationinduced meningiomas after an interval of 22 years and briefly discuss 16 previous reports on this occurrence in benign astrocytomas