http://www.recercat.cat:80/handle/2072/48843 Sun, 26 May 2013 07:50:42 GMT 2013-05-26T07:50:42Z http://www.recercat.cat:80/bitstream/id/34158/ http://www.recercat.cat:80/handle/2072/48843 http://www.recercat.cat:80/handle/2072/211161 Fine-Tuning Translation Kinetics Selection as the Driving Force of Codon Usage Bias in the Hepatitis A Virus Capsid Aragonès, Ll.; Guix Arnau, Susana; Ribes Mora, Enric; Bosch, Albert; Pintó Solé, Rosa María Hepatitis A virus (HAV), the prototype of genus Hepatovirus, has several unique biological characteristics that distinguish it from other members of the Picornaviridae family. Among these, the need for an intact eIF4G factor for the initiation of translation results in an inability to shut down host protein synthesis by a mechanism similar to that of other picornaviruses. Consequently, HAV must inefficiently compete for the cellular translational machinery and this may explain its poor growth in cell culture. In this context of virus/cell competition, HAV has strategically adopted a naturally highly deoptimized codon usage with respect to that of its cellular host. With the aim to optimize its codon usage the virus was adapted to propagate in cells with impaired protein synthesis, in order to make tRNA pools more available for the virus. A significant loss of fitness was the immediate response to the adaptation process that was, however, later on recovered and more associated to a re-deoptimization rather than to an optimization of the codon usage specifically in the capsid coding region. These results exclude translation selection and instead suggest fine-tuning translation kinetics selection as the underlying mechanism of the codon usage bias in this specific genome region. Additionally, the results provide clear evidence of the Red Queen dynamics of evolution since the virus has very much evolved to re-adapt its codon usage to the environmental cellular changing conditions in order to recover the original fitness. http://www.recercat.cat:80/handle/2072/211161 http://www.recercat.cat:80/handle/2072/211081 Inhibition of cellular protein secretion by norwalk virus nonstructural protein p22 requires a mimic of an endoplasmic reticulum export signal. Sharp. T.M.; Guix Arnau, Susana; Katayama, K.; Crawford, S.E.; Estes, M.K. Protein trafficking between the endoplasmic reticulum (ER) and Golgi apparatus is central to cellular homeostasis. ER export signals are utilized by a subset of proteins to rapidly exit the ER by direct uptake into COPII vesicles for transport to the Golgi. Norwalk virus nonstructural protein p22 contains a YXΦESDG motif that mimics a di-acidic ER export signal in both sequence and function. However, unlike normal ER export signals, the ER export signal mimic of p22 is necessary for apparent inhibition of normal COPII vesicle trafficking, which leads to Golgi disassembly and antagonism of Golgi-dependent cellular protein secretion. This is the first reported function for p22. Disassembly of the Golgi apparatus was also observed in cells replicating Norwalk virus, which may contribute to pathogenesis by interfering with cellular processes that are dependent on an intact secretory pathway. These results indicate that the ER export signal mimic is critical to the antagonistic function of p22, shown herein to be a novel antagonist of ER/Golgi trafficking. This unique and well-conserved human norovirus motif is therefore an appealing target for antiviral drug development. http://www.recercat.cat:80/handle/2072/211081 http://www.recercat.cat:80/handle/2072/211032 Characterizing RecA-Independent Induction of Shiga toxin2-encoding Phages by EDTA Treatment Imamovic, L.; Muniesa Pérez, Ma Teresa The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA. http://www.recercat.cat:80/handle/2072/211032 http://www.recercat.cat:80/handle/2072/210885 Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS Baños Molina, Rosa Carmen; Vivero, A.; Aznar Vicente, S.; García, J.; Pons Vallès, Miquel; Madrid Xufré, Cristina; Juárez Giménez, Antonio Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization. http://www.recercat.cat:80/handle/2072/210885 http://www.recercat.cat:80/handle/2072/210815 Aeromonas hydrophila lateral flagella gene transcriptional hierarchy Wilhelms, M.; González, V.; Tomàs Magaña, Juan; Merino Montero, Susana Aeromonas hydrophila AH-3 lateral flagella are not assembled when bacteria grow in liquid media; however, lateral flagellar genes are transcribed. Our results indicate that A. hydrophila lateral flagellar genes are transcribed at three levels (class I to III genes) and share some similarities with, but have many important differences from, genes of Vibrio parahaemolyticus. A. hydrophila lateral flagellum class I gene transcription is σ(70) dependent, which is consistent with the fact that lateral flagellum is constitutively transcribed, in contrast to the characteristics of V. parahaemolyticus. The fact that multiple genes are included in class I highlights that lateral flagellar genes are less hierarchically transcribed than polar flagellum genes. The A. hydrophila lafK-fliEJL gene cluster (where the subscript L distinguishes genes for lateral flagella from those for polar flagella) is exclusively from class I and is in V. parahaemolyticus class I and II. Furthermore, the A. hydrophila flgAMNL cluster is not transcribed from the σ(54)/LafK-dependent promoter and does not contain class II genes. Here, we propose a gene transcriptional hierarchy for the A. hydrophila lateral flagella. http://www.recercat.cat:80/handle/2072/210815 http://www.recercat.cat:80/handle/2072/210767 Experimental Identification of Actinobacillus pleuropneumoniae Strains L20 and JL03 Heptosyltransferases, Evidence for a New Heptosyltransferase Signature Sequence. Merino Montero, Susana; Knirel, Y.A.; Regué Queralt, Miguel, 1953-; Tomàs Magaña, Juan We experimentally identified the activities of six predicted heptosyltransferases in Actinobacillus pleuropneumoniae genome serotype 5b strain L20 and serotype 3 strain JL03. The initial identification was based on a bioinformatic analysis of the amino acid similarity between these putative heptosyltrasferases with others of known function from enteric bacteria and Aeromonas. The putative functions of all the Actinobacillus pleuropneumoniae heptosyltrasferases were determined by using surrogate LPS acceptor molecules from well-defined A. hydrophyla AH-3 and A. salmonicida A450 mutants. Our results show that heptosyltransferases APL_0981 and APJL_1001 are responsible for the transfer of the terminal outer core D-glycero-D-manno-heptose (D,D-Hep) residue although they are not currently included in the CAZY glycosyltransferase 9 family. The WahF heptosyltransferase group signature sequence [S(T/S)(GA)XXH] differs from the heptosyltransferases consensus signature sequence [D(TS)(GA)XXH], because of the substitution of D(261) for S(261), being unique. http://www.recercat.cat:80/handle/2072/210767 http://www.recercat.cat:80/handle/2072/210681 Melanization and pathogenicity in Tenebrio molitor and Pacifastacus leniusculus by Aeromonas hydrophila. Noonin, C.; Jiravanichpaisal, P.; Söderhäll, I.; Merino Montero, Susana; Tomàs Magaña, Juan; Söderhäll1, K. Aeromonas hydrophila is the most common Aeromonas species causing infections in human and other animals such as amphibians, reptiles, fish and crustaceans. Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Several mutant strains of A. hydrophila AH-3 were initially used to study their virulence in two animal species, Pacifastacus leniusculus (crayfish) and Tenebrio molitor larvae (mealworm). The AH-3 strains used in this study have mutations in genes involving the synthesis of flagella, LPS structures, secretion systems, and some other factors, which have been reported to be involved in A. hydrophila pathogenicity. Our study shows that the LPS (O-antigen and external core) is the most determinant A. hydrophila AH-3 virulence factor in both animals. Furthermore, we studied the immune responses of these hosts to infection of virulent or non-virulent strains of A. hydrophila AH-3. The AH-3 wild type (WT) containing the complete LPS core is highly virulent and this bacterium strongly stimulated the prophenoloxidase activating system resulting in melanization in both crayfish and mealworm. In contrast, the ΔwaaE mutant which has LPS without O-antigen and external core was non-virulent and lost ability to stimulate this system and melanization in these two animals. The high phenoloxidase activity found in WT infected crayfish appears to result from a low expression of pacifastin, a prophenoloxidase activating enzyme inhibitor, and this gene expression was not changed in the ΔwaaE mutant infected animal and consequently phenoloxidase activity was not altered as compared to non-infected animals. Therefore we show that the virulence factors of A. hydrophila are the same regardless whether an insect or a crustacean is infected and the O-antigen and external core is essential for activation of the proPO system and as virulence factors for this bacterium. http://www.recercat.cat:80/handle/2072/210681 http://www.recercat.cat:80/handle/2072/210376 Type 1 fimbriae, a colonization factor of uropathogenic Escherichia coli, are controlled by the metabolic sensor CRP-cAMP. Müller, C.M.; Aberg, A.; Straseviciene, J.; Emödy, L.; Uhlin, B.E.; Balsalobre Parra, Carlos Type 1 fimbriae are a crucial factor for the virulence of uropathogenic Escherichia coli during the first steps of infection by mediating adhesion to epithelial cells. They are also required for the consequent colonization of the tissues and for invasion of the uroepithelium. Here, we studied the role of the specialized signal transduction system CRP-cAMP in the regulation of type 1 fimbriation. Although initially discovered by regulating carbohydrate metabolism, the CRP-cAMP complex controls a major regulatory network in Gram-negative bacteria, including a broad subset of genes spread into different functional categories of the cell. Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation. The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity. Moreover, the underlying studies revealed that CRP-cAMP controls the expression of another global regulator in Gram-negative bacteria, the leucine-responsive protein Lrp. CRP-cAMP-mediated repression is limiting the switch from the non-fimbriated to the fimbriated state. Consistently, a drop in the intracellular concentration of cAMP due to altered physiological conditions (e.g. growth in presence of glucose) increases the percentage of fimbriated cells in the bacterial population. We also provide evidence that the repression of type 1 fimbriae by CRP-cAMP occurs during fast growth conditions (logarithmic phase) and is alleviated during slow growth (stationary phase), which is consistent with an involvement of type 1 fimbriae in the adaptation to stress conditions by promoting biofilm growth or entry into host cells. Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues. http://www.recercat.cat:80/handle/2072/210376 http://www.recercat.cat:80/handle/2072/209578 Antibiotics shaping bacterial genome: deletion of an IS91 flanked virulence determinant upon exposure to suinhibitory antibiotic concentrations Pedró, Laura; Baños Molina, Rosa Carmen; Aznar Vicente, S.; Madrid Xufré, Cristina; Balsalobre Parra, Carlos; Juárez Giménez, Antonio The nucleoid-associated proteins Hha and YdgT repress the expression of the toxin α-hemolysin. An Escherichia coli mutant lacking these proteins overexpresses the toxin α-hemolysin encoded in the multicopy recombinant plasmid pANN202-312R. Unexpectedly, we could observe that this mutant generated clones that no further produced hemolysin (Hly-). Generation of Hly- clones was dependent upon the presence in the culture medium of the antibiotic kanamycin (km), a marker of the hha allele (hha::Tn5). Detailed analysis of different Hly- clones evidenced that recombination between partial IS91 sequences that flank the hly operon had occurred. A fluctuation test evidenced that the presence of km in the culture medium was underlying the generation of these clones. A decrease of the km concentration from 25 mg/l to 12.5 mg/l abolished the appearance of Hly- derivatives. We considered as a working hypothesis that, when producing high levels of the toxin (combination of the hha ydgT mutations with the presence of the multicopy hemolytic plasmid pANN202-312R), the concentration of km of 25 mg/l resulted subinhibitory and stimulated the recombination between adjacent IS91 flanking sequences. To further test this hypothesis, we analyzed the effect of subinhibitory km concentrations in the wild type E. coli strain MG1655 harboring the parental low copy number plasmid pHly152. At a km concentration of 5 mg/l, subinhibitory for strain MG1655 (pHly152), generation of Hly- clones could be readily detected. Similar results were also obtained when, instead of km, ampicillin was used. IS91 is flanking several virulence determinants in different enteric bacterial pathogenic strains from E. coli and Shigella. The results presented here evidence that stress generated by exposure to subinhibitory antibiotic concentrations may result in rearrangements of the bacterial genome. Whereas some of these rearrangements may be deleterious, others may generate genotypes with increased virulence, which may resume infection. http://www.recercat.cat:80/handle/2072/209578 http://www.recercat.cat:80/handle/2072/209577 Similar and Divergent Effects of ppGpp and DksA Deficiencies on Transcription in Escherichia coli. Aberg, A.; Fernández Vázquez, Jorge; Cabrer-Panes, J.D.; Sànchez, Àlex (Sànchez Pla); Balsalobre Parra, Carlos The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp0) and/or DksA ( dksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksAdeficient strains, respectively, increasing to 13% of all genes in the ppGpp0 dksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli. http://www.recercat.cat:80/handle/2072/209577 http://www.recercat.cat:80/handle/2072/208108 La Genetica forense: utilidad en la administracion de Justicia, repercusion social y aspectos eticos Mestres i Naval, Francesc; Vives-Rego, José Los diccionarios oficiales de la Real Academia de Lengua Española no contienen la palabra forensia, que sin embargo es de amplio uso en el entorno jurídico. El término"forensia" se usa en la mayoría de los casos como equivalente a"ciencia forense" significando la aplicación de la ciencia (en su sentido más amplio) para responder a cuestiones legales y ayudar a la administración de la Justicia. Desde un punto de vista práctico la forensia se utiliza como una vía de autentificación de datos y hechos que tienen interés legal. El uso moderno del término"forensia" en vez de"ciencia forense" es considerado incorrecto por ciertos sectores de la jurisprudencia básicamente por dos motivos: i) la forensia en sentido amplio también implica campos no científicos como el arte y la misma jurisprudencia y ii) por ser el término"forensia" un sinónimo de legal o relacionado con los tribunales. http://www.recercat.cat:80/handle/2072/208108 http://www.recercat.cat:80/handle/2072/206261 Quorum-sensing regulates biofilm formation in Vibrio scophthalmi García Aljaro, Cristina; Melado Rovira, Silvia; Milton, Debra L.; Blanch i Gisbert, Anicet Background: In a previous study, we demonstrated that Vibrio scophthalmi, the most abundant Vibrio species among the marine aerobic or facultatively anaerobic bacteria inhabiting the intestinal tract of healthy cultured turbot (Scophthalmus maximus), contains at least two quorum-sensing circuits involving two types of signal molecules (a 3-hydroxy-dodecanoyl-homoserine lactone and the universal autoinducer 2 encoded by luxS). The purpose of this study was to investigate the functions regulated by these quorum sensing circuits in this vibrio by constructing mutants for the genes involved in these circuits. Results. The presence of a homologue to the Vibrio harveyi luxR gene encoding a main transcriptional regulator, whose expression is modulated by quorum&br&sensing signal molecules in other vibrios, was detected and sequenced. The V. scophthalmi LuxR protein displayed a maximum amino acid identity of 82% with SmcR, the LuxR homologue found in Vibrio vulnificus. luxR and luxS null mutants were constructed and their phenotype analysed. Both mutants displayed reduced biofilm formation in vitro as well as differences in membrane protein expression by mass-spectrometry analysis. Additionally, a recombinant strain of V. scophthalmi carrying the lactonase AiiA from Bacillus cereus, which causes hydrolysis of acyl homoserine lactones, was included in the study. Conclusions: V. scophthalmi shares two quorum sensing circuits, including the main transcriptional regulator luxR, with some pathogenic vibrios such as V. harveyi and V. anguillarum. However, contrary to these pathogenic vibrios no virulence factors (such as protease production) were found to be quorum sensing regulated in this bacterium. Noteworthy, biofilm formation was altered in luxS and luxR mutants. In these mutants a different expression profile of membrane proteins were observed with respect to the wild type strain suggesting that quorum sensing could play a role in the regulation of the adhesion mechanisms of this bacterium. http://www.recercat.cat:80/handle/2072/206261 http://www.recercat.cat:80/handle/2072/202654 Estudio fisico-quimico y microbiologico de la fermentacion de aceitunas verdes arbequinas Torre, J. E de la; Moya, E. R.; Bota Prieto, Enric; Sancho Valls, J.(Josep) A pesar del creciente consumo, existe poca información disponible sobre las aceitunas verdes en salmuera, sobre todo de la variedad Arbequina. Con este producto se suelen presentar problemas de conservación debido a su baja acidez y valores relativamente altos de pH. Se ha realizado un estudio comparativo, con aceituna Arbequina, de fermentación espontánea y de fermentación dirigida utilizando cepas de bacterias lácticas, aisladas de muestras comercializadas de estas olivas, que se han identificado en todos los casos como Lactobacillus plantarum. En el presente trabajo se ofrecen los resultados obtenidos en la determinación de los principales parámetros físico-químicos y microbiológicos de las aguas de lavado de las aceitunas y del caldo de fermentación a lo largo del proceso. A partir de los resultados expuestos se proponen algunos cambios significativos en la técnica usual para la fermentación de la aceituna verde Arbequina. http://www.recercat.cat:80/handle/2072/202654 http://www.recercat.cat:80/handle/2072/182758 Raw sewage harbors diverse viral populations Cantalupo, Paul G.; Calgua, Byron; Zhao, Guoyan; Hundesa Gonfa, Ayalkibet; Wier, Adam D.; Katz, Josh P.; Grabe, Michael; Hendrix, Roger W.; Gironès Llop, Rosina; Wang, David; Pipasa, James M. At this time, about 3,000 different viruses are recognized, but metagenomic studies suggest that these viruses are a small fraction of the viruses that exist in nature. We have explored viral diversity by deep sequencing nucleic acids obtained from virion populations enriched from raw sewage. We identified 234 known viruses, including 17 that infect humans. Plant, insect, and algal viruses as well as bacteriophages were also present. These viruses represented 26 taxonomic families and included viruses with single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), positive-sense ssRNA [ssRNA(¿)], and dsRNA genomes. Novel viruses that could be placed in specific taxa represented 51 different families, making untreated wastewater the most diverse viral metagenome (genetic material recovered directly from environmental samples) examined thus far. However, the vast majority of sequence reads bore little or no sequence relation to known viruses and thus could not be placed into specific taxa. These results show that the vast majority of the viruses on Earth have not yet been characterized. Untreated wastewater provides a rich matrix for identifying novel viruses and for studying virus diversity. http://www.recercat.cat:80/handle/2072/182758 http://www.recercat.cat:80/handle/2072/182389 The evolution of microbial life: paradigm changes in microbiology. Guerrero, Ricardo, 1943-; Berlanga Herranz, Mercedes http://www.recercat.cat:80/handle/2072/182389 http://www.recercat.cat:80/handle/2072/182388 Professor Joan Oró (1923-2004). Biography and bibliography Guerrero, Ricardo, 1943- http://www.recercat.cat:80/handle/2072/182388 http://www.recercat.cat:80/handle/2072/179918 Indirect DNA Readout by an H-NS Related Protein: Structure of the DNA Complex of the C-Terminal Domain of Ler Cordeiro, Tiago N.; Schmidt, Holger; Madrid Xufré, Cristina; Juárez Giménez, Antonio; Bernadó i Peretó, Pau; Griesinger, Christian; García, Jesús; Pons Vallès, Miquel Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family. http://www.recercat.cat:80/handle/2072/179918 http://www.recercat.cat:80/handle/2072/179382 Isolation of a novel monkey adenovirus reveals a new phylogenetic clade in the evolutionary history of simian adenoviruses Maluquer de Motes i Porta, Carles; Hundesa Gonfa, Ayalkibet; Almeida, Francisca C.; Bofill Mas, Sílvia; Gironès Llop, Rosina Adenoviruses of primates include human (HAdV) and simian (SAdV) isolates classified into 8 species (Human Adenovirus A to G, and Simian Adenovirus A). In this study, a novel adenovirus was isolated from a colony of cynomolgus macaques (Macaca fascicularis) and subcultured in VERO cells. Its complete genome was purified and a region encompassing the hexon gene, the protease gene, the DNA binding protein (DBP) and the 100 kDa protein was amplified by PCR and sequenced by primer walking. Sequence analysis of these four genes showed that the new isolate had 80% identity to other primate adenoviruses and lacked recombination events. The study of the evolutionary relationships of this new monkey AdV based on the combined sequences of the four genes supported a close relationship to SAdV-3 and SAdV-6, lineages isolated from Rhesus monkeys. The clade formed by these three types is separated from the remaining clades and establishes a novel branch that is related to species HAdV-A, F and G. However, the genetic distance corresponding to the newly isolated monkey AdV considerably differs from these as to belong to a new, not yet established species. Results presented here widen our knowledge on SAdV and represents an important contribution to the understanding of the evolutionary history of primate adenoviruses. http://www.recercat.cat:80/handle/2072/179382 http://www.recercat.cat:80/handle/2072/179381 Antibiotic resistance genes in the bacteriophage DNA fraction of environmental samples Colomer-Lluch, Marta; Jofre i Torroella, Joan; Muniesa Pérez, Ma Teresa Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, ß-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to ß-lactam antibiotics is conferred by ß-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to ß-lactam antibiotics, namely two ß-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment. http://www.recercat.cat:80/handle/2072/179381 http://www.recercat.cat:80/handle/2072/117268 Surveillance of adenoviruses and noroviruses in European recreational waters Wyn-Jones, A. Peter; Carducci, Annalaura; Cook, Nigel; D'Agostino, Martin; Divizia, Maurizio; Fleischer, Jens; Gantzer, Christophe; Gawler, Andrew; Gironès Llop, Rosina; Höller, Christiane; Roda Husman, Ana Maria de; Kozyra, Iwona; López-Pila, Juan; Muscillo, Michele; Nascimento, Maria; Papageorgiou, George; Rutjes, Saskia; Sellwood, Jane; Wyer, Mark; Kay, David Exposure to human pathogenic viruses in recreational waters has been shown to cause disease outbreaks. In the context of Article 14 of the revised European Bathing Waters Directive 2006/7/EC (rBWD, CEU, 2006) a Europe-wide surveillance study was carried out to determine the frequency of occurrence of two human enteric viruses in recreational waters. Adenoviruses were selected based on their near-universal shedding and environmental survival, and noroviruses (NoV) selected as being the most prevalent gastroenteritis agent worldwide. Concentration of marine and freshwater samples was done by adsorption/elution followed by molecular detection by (RT)-PCR. Out of 1410 samples, 553 (39.2%) were positive for one or more of the target viruses. Adenoviruses, detected in 36.4% of samples, were more prevalent than noroviruses (9.4%), with 3.5% GI and 6.2% GII, some samples being positive for both GI and GII. Of 513 human adenovirus-positive samples, 63 (12.3%) were also norovirus-positive, whereas 69 (7.7%) norovirus-positive samples were adenovirus-negative. More freshwater samples than marine water samples were virus-positive. Out of a small selection of samples tested for adenovirus infectivity, approximately one-quarter were positive. Sixty percent of 132 nested-PCR adenovirus-positive samples analysed by quantitative PCR gave a mean value of over 3000 genome copies per L of water. The simultaneous detection of infectious adenovirus and of adenovirus and NoV by (RT)PCR suggests that the presence of infectious viruses in recreational waters may constitute a public health risk upon exposure. These studies support the case for considering adenoviruses as an indicator of bathing water quality. http://www.recercat.cat:80/handle/2072/117268 http://www.recercat.cat:80/handle/2072/117267 Quantification of human adenoviruses in European recreational waters Bofill Mas, Sílvia; Calgua, Byron; Clemente Casares, Pilar; La Rosa, Giuseppina; Iaconelli, Marcello; Muscillo, Michele; Rutjes, Saskia; Roda Husman, Ana Maria de; Grünert, Andreas; Graver, Ingeburg; Verani, Marco; Carducci, Annalaura; Calvo, Miquel; Wyn-Jones, A. Peter; Gironès Llop, Rosina The presence of human adenoviruses in recreational water might cause disease in the population upon exposure. Human adenoviruses detected by PCR could also serve as indicators of the virological water quality. In order to assess the applicability of human adenoviruses to the evaluation of the faecal contamination in European bathing waters, a real-time quantitative PCR assay was developed for the quantification of human adenoviruses in 132 samples collected from 24 different recreational marine and freshwater sites in nine European countries.Selected samples presenting positive nested-PCR results for human adenoviruses were analyzed using quantitative PCR and 80 samples from a total of 132 produced quantitative results with mean values of 3.2x102 10 per 100 ml of water, human adenovirus 41 being the most prevalent serotype. Human adenoviruses were quantified in samples from all 15 surveillance laboratories. Statistical analysis showed no homogeneous linear relation between humanadenoviruses and E. coli, intestinal enterococci or somatic coliphages concentrations in the tested samples when considering all the data together. Significant correlations between human adenoviruses and at least one of the other indicators were observed only when data from individual Laboratories were considered. The quantification of human adenoviruses may provide complementary information in relation to the use of bacterial standards in the control of water quality in bathing water. http://www.recercat.cat:80/handle/2072/117267 http://www.recercat.cat:80/handle/2072/117266 Development and application of a one-step low cost procedure to concentrate viruses from seawater samples Calgua, Byron; Mengewein, A.; Grünert, Andreas; Bofill Mas, Sílvia; Clemente Casares, Pilar; Hundesa Gonfa, Ayalkibet; Wyn-Jones, A. Peter; López-Pila, Juan; Gironès Llop, Rosina A novel and simple procedure for concentrating adenoviruses from seawater samples is described. The technique entails the adsorption of viruses to pre-flocculated skimmed milk proteins, allowing the flocs to sediment by gravity, and dissolving the separated sediment in phosphate buffer. Concentrated virus may be detected by PCR techniques following nucleic acid extraction. The method requires no specialized equipment other than that usually available in routine public health laboratories, and due to its straightforwardness it allows the processing of a larger number of water samples simultaneously. The usefulness of the method was demonstrated in concentration of virus in multiple seawater samples during a survey of adenoviruses in coastal waters. http://www.recercat.cat:80/handle/2072/117266 http://www.recercat.cat:80/handle/2072/83818 Newly described human polyomaviruses Merkel Cell, KI and WU are present in urban sewage and may represent potential environmental contaminants Bofill Mas, Sílvia; Rodríguez-Manzano, Jesús; Calgua, Byron; Carratalà, Anna; Gironès Llop, Rosina Recently, three new polyomaviruses (KI, WU and Merkel cell polyomavirus) have been reported to infect humans. It has also been suggested that lymphotropic polyomavirus, a virus of simian origin, infects humans. KI and WU polyomaviruses have been detected mainly in specimens from the respiratory tract while Merkel cell polyomavirus has been described in a very high percentage of Merkel cell carcinomas. The distribution, excretion level and transmission routes of these viruses remain unknown. Here we analyzed the presence and characteristics of newly described human polyomaviruses in urban sewage and river water in order to assess the excretion level and the potential role of water as a route of transmission of these viruses. Nested-PCR assays were designed for the sensitive detection of the viruses studied and the amplicons obtained were confirmed by sequencing analysis. The viruses were concentrated following a methodology previously developed for the detection of JC and BK human polyomaviruses in environmental samples. JC polyomavirus and human adenoviruses were used as markers of human contamination in the samples. Merkel cell polyomavirus was detected in 7/8 urban sewage samples collected and in 2/7 river water samples. Also one urine sample from a pregnant woman, out of 4 samples analyzed, was positive for this virus. KI and WU polyomaviruses were identified in 1/8 and 2/8 sewage samples respectively. The viral strains detected were highly homologous with other strains reported from several other geographical areas. Lymphotropic polyomavirus was not detected in any of the 13 sewage neither in 9 biosolid/sludge samples analyzed. This is the first description of a virus isolated from sewage and river water with a strong association with cancer. Our data indicate that the Merkel cell polyomavirus is prevalent in the population and that it may be disseminated through the fecal/urine contamination of water. The procedure developed may constitute a useful tool for studying the excreted strains, prevalence and transmission of these recently described polyomaviruses. http://www.recercat.cat:80/handle/2072/83818 http://www.recercat.cat:80/handle/2072/49480 Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes Serra Moreno, Ruth; Acosta, Sandra; Hernalsteens, Jean Pierre; Jofre Torroella, Joan; Muniesa Pérez, Ma Teresa Background: The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene. Results: Seven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol. Conclusion: This approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer. http://www.recercat.cat:80/handle/2072/49480