RESUMEN
Integrons are genetic elements that incorporate mobile gene cassettes by site-specific recombination and express them as an operon from a promoter (Pc) located upstream of the cassette insertion site. Most gene cassettes found in integrons contain only one gene followed by an attC recombination site. We have recently shown that a specific lineage of group IIC introns, named group IIC-attC introns, inserts into the bottom strand sequence of attC sites. Here, we show that S.ma.I2, a group IIC-attC intron inserted in an integron cassette array of Serratia marcescens, impedes transcription from Pc while allowing expression of the following antibiotic resistance cassette using an internal outward-oriented promoter (P(out)). Bioinformatic analyses indicate that one or two putative P(out), which have sequence similarities with the Escherichia coli consensus promoters, are conserved in most group IIC-attC intron sequences. We show that P(out) with different versions of the -35 and -10 sequences are functionally active in expressing a promoterless chloramphenicol acetyltransferase (cat) reporter gene in E. coli. P(out) in group IIC-attC introns may therefore play a role in the expression of one or more gene cassettes whose transcription from Pc would otherwise be impeded by insertion of the intron.
Asunto(s)
Integrones , Intrones , Regiones Promotoras Genéticas , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/análisis , Biología Computacional , Genes Bacterianos , Genes Reporteros , Datos de Secuencia Molecular , Serratia marcescens/genética , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
Bacterial group IIC introns are a subclass of group II intron ribozymes that are typically located downstream from transcriptional terminators. Class IIC-attC introns constitute a monophyletic subset of subgroup IIC, which preferentially insert into site-specific recombination sequences for integron integrases (attC). attCs are a diverse family of nucleotide sequences composed of conserved inverted repeats that flank a variable, but palindromic, central region. In this study, we used both PCR and colony patch hybridization methods to determine the basis for recognition of the attC(aadA1) stem-loop motif by the Serratia marcescens intron (S.ma.I2) in vivo. The quantitative results showed that mobility into the wild-type site occurs at a frequency of 18%, and is strongly biased by the orientation of the homing site relative to the direction of DNA replication. S.ma.I2 mobility results into mutant attC(aadA1) sites are consistent with recognition of stem-loop motifs in unwound DNA. The homing frequency results showed that, while the entire attC sequence is not necessary for recognition of the insertion site, short deletions of the attC stem-loop motif inhibited the intron mobility. Moreover, our data show that S.ma.I2 requires a bulged base in the folded attC stem for high homing frequency. We demonstrate that the IBS1/IBS3 motifs and two bulge bases conserved among attCs determine S.ma.I2 homing specificity for the attC bottom strand. These results suggest that class IIC-attC introns tolerate attC variation by recognition of a bulged hairpin DNA motif rather than a specific sequence.
Asunto(s)
Integrones/genética , Intrones , Secuencia de Bases , Sitios de Unión/genética , Replicación del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Integrasas/genética , Integrasas/metabolismo , Secuencias Invertidas Repetidas , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis , Conformación de Ácido Nucleico , ARN Catalítico/genética , ARN Catalítico/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , Serratia marcescens/genética , Serratia marcescens/metabolismoRESUMEN
Integrons are natural expression vectors in which gene cassettes are integrated downstream of a promoter region by a site-specific recombinase. Gene cassettes usually consist of a single gene followed by a recombination site designated attC. A major unanswered question is how a gene becomes associated with an attC site. Here, we investigate the potential role of a specific lineage of group IIC introns, named group IIC-attC, in cassette formation. Group IIC-attC introns preferentially target attC while retaining the ability to target transcriptional terminators. We show using a PCR-based mobility assay with Escherichia coli that the S.ma.I2 intron from the genome of a clinical isolate of Serratia marcescens can target both attC site and putative terminator motifs of resistance genes. Quantitative results showed that S.ma.I2 is more efficient in targeting various attC sequences than three group IIC-attC introns (54 to 64% sequence identity) from the genomes of environmental isolates. We also show that purified group IIC-attC intron-encoded reverse transcriptases have both RNA-dependent and DNA-dependent DNA polymerase activities in vitro. These data permit us to suggest a new model for gene cassette formation, in which a group IIC-attC intron targets separately a transcriptional terminator adjoining a gene and an isolated attC, joins the gene and the attC by homologous recombination, and then splices and reverse transcribes a gene-attC RNA template, leading to the formation of a cassette.
Asunto(s)
ADN Bacteriano/genética , Integrones/genética , Intrones/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/genética , Modelos Genéticos , Reacción en Cadena de la Polimerasa , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Serratia marcescens/genéticaRESUMEN
BACKGROUND: Mobilizing research evidence for daily decision-making is challenging for health system decision-makers. In a previous qualitative paper, we showed the current mix of supports that Canadian health-care organizations have in place and the ones that are perceived to be helpful to facilitate the use of research evidence in health system decision-making. Factors influencing the implementation of such supports remain poorly described in the literature. Identifying the barriers to and facilitators of different interventions is essential for implementation of effective, context-specific, supports for evidence-informed decision-making (EIDM) in health systems. The purpose of this study was to identify (a) barriers and facilitators to implementing supports for EIDM in Canadian health-care organizations, (b) views about emerging development of supports for EIDM, and (c) views about the priorities to bridge the gaps in the current mix of supports that these organizations have in place. METHODS: This qualitative study was conducted in three types of health-care organizations (regional health authorities, hospitals, and primary care practices) in two Canadian provinces (Ontario and Quebec). Fifty-seven in-depth semi-structured telephone interviews were conducted with senior managers, library managers, and knowledge brokers from health-care organizations that have already undertaken strategic initiatives in knowledge translation. The interviews were taped, transcribed, and then analyzed thematically using NVivo 9 qualitative data analysis software. RESULTS: Limited resources (i.e., money or staff), time constraints, and negative attitudes (or resistance) toward change were the most frequently identified barriers to implementing supports for EIDM. Genuine interest from health system decision-makers, notably their willingness to invest money and resources and to create a knowledge translation culture over time in health-care organizations, was the most frequently identified facilitator to implementing supports for EIDM. The most frequently cited views about emerging development of supports for EIDM were implementing accessible and efficient systems to support the use of research in decision-making (e.g., documentation and reporting tools, communication tools, and decision support tools) and developing and implementing an infrastructure or position where the accountability for encouraging knowledge use lies. The most frequently stated priorities for bridging the gaps in the current mix of supports that these organizations have in place were implementing technical infrastructures to support research use and to ensure access to research evidence and establishing formal or informal ties to researchers and knowledge brokers outside the organization who can assist in EIDM. CONCLUSIONS: These results provide insights on the type of practical implementation imperatives involved in supporting EIDM.
Asunto(s)
Técnicas de Apoyo para la Decisión , Práctica Clínica Basada en la Evidencia/estadística & datos numéricos , Política de Salud , Servicios de Salud , Investigación Biomédica Traslacional , Actitud del Personal de Salud , Difusión de Innovaciones , Prioridades en Salud , Humanos , Ontario , Innovación Organizacional , Organizaciones/estadística & datos numéricos , QuebecRESUMEN
BACKGROUND: This protocol builds on the development of a) a framework that identified the various supports (i.e. positions, activities, interventions) that a healthcare organisation or health system can implement for evidence-informed decision-making (EIDM) and b) a qualitative study that showed the current mix of supports that some Canadian healthcare organisations have in place and the ones that are perceived to facilitate the use of research evidence in decision-making. Based on these findings, we developed a web survey to collect cross-sectional data about the specific supports that regional health authorities and hospitals in two Canadian provinces (Ontario and Quebec) have in place to facilitate EIDM. METHODS/DESIGN: This paper describes the methods for a cross-sectional web survey among 32 regional health authorities and 253 hospitals in the provinces of Quebec and Ontario (Canada) to collect data on the current mix of organisational supports that these organisations have in place to facilitate evidence-informed decision-making. The data will be obtained through a two-step survey design: a 10-min survey among CEOs to identify key units and individuals in regard to our objectives (step 1) and a 20-min survey among managers of the key units identified in step 1 to collect information about the activities performed by their unit regarding the acquisition, assessment, adaptation and/or dissemination of research evidence in decision-making (step 2). The study will target three types of informants: CEOs, library/documentation centre managers and all other key managers whose unit is involved in the acquisition, assessment, adaptation/packaging and/or dissemination of research evidence in decision-making. We developed an innovative data collection system to increase the likelihood that only the best-informed respondent available answers each survey question. The reporting of the results will be done using descriptive statistics of supports by organisation type and by province. DISCUSSION: This study will be the first to collect and report large-scale cross-sectional data on the current mix of supports health system organisations in the two most populous Canadian provinces have in place for evidence-informed decision-making. The study will also provide useful information to researchers on how to collect organisation-level data with reduced risk of self-reporting bias.
Asunto(s)
Medicina Basada en la Evidencia , Instituciones de Salud/estadística & datos numéricos , Organizaciones/estadística & datos numéricos , Formulación de Políticas , Estudios Transversales , Sistemas de Apoyo a Decisiones Clínicas/estadística & datos numéricos , Política de Salud , Humanos , Ontario , Quebec , Programas Médicos Regionales/organización & administración , Programas Médicos Regionales/estadística & datos numéricosRESUMEN
BACKGROUND: Decisions regarding health systems are sometimes made without the input of timely and reliable evidence, leading to less than optimal health outcomes. Healthcare organizations can implement tools and infrastructures to support the use of research evidence to inform decision-making. OBJECTIVES: The purpose of this study was to profile the supports and instruments (i.e., programs, interventions, instruments or tools) that healthcare organizations currently have in place and which ones were perceived to facilitate evidence-informed decision-making. METHODS: In-depth semi-structured telephone interviews were conducted with individuals in three different types of positions (i.e., a senior management team member, a library manager, and a 'knowledge broker') in three types of healthcare organizations (i.e., regional health authorities, hospitals and primary care practices) in two Canadian provinces (i.e., Ontario and Quebec). The interviews were taped, transcribed, and then analyzed thematically using NVivo 9 qualitative data analysis software. RESULTS: A total of 57 interviews were conducted in 25 organizations in Ontario and Quebec. The main findings suggest that, for the healthcare organizations that participated in this study, the following supports facilitate evidence-informed decision-making: facilitating roles that actively promote research use within the organization; establishing ties to researchers and opinion leaders outside the organization; a technical infrastructure that provides access to research evidence, such as databases; and provision and participation in training programs to enhance staff's capacity building. CONCLUSIONS: This study identified the need for having a receptive climate, which laid the foundation for the implementation of other tangible initiatives and supported the use of research in decision-making. This study adds to the literature on organizational efforts that can increase the use of research evidence in decision-making. Some of the identified supports may increase the use of research evidence by decision-makers, which may then lead to more informed decisions, and hopefully to a strengthened health system and improved health.
Asunto(s)
Toma de Decisiones , Atención a la Salud/organización & administración , Organizaciones/organización & administración , Técnicas de Apoyo para la Decisión , Medicina Basada en la Evidencia , Humanos , Ontario , Quebec , Regionalización/organización & administraciónRESUMEN
BACKGROUND: Evidence-informed health policymaking logically depends on timely access to research evidence. To our knowledge, despite the substantial political and societal pressure to enhance the use of the best available research evidence in public health policy and program decision making, there is no study addressing availability of peer-reviewed research in Canadian health ministries. OBJECTIVES: To assess availability of (1) a purposive sample of high-ranking scientific journals, (2) bibliographic databases, and (3) health library services in the fourteen Canadian health ministries. METHODS: From May to October 2011, we conducted a cross-sectional survey among librarians employed by Canadian health ministries to collect information relative to availability of scientific journals, bibliographic databases, and health library services. Availability of scientific journals in each ministry was determined using a sample of 48 journals selected from the 2009 Journal Citation Reports (Sciences and Social Sciences Editions). Selection criteria were: relevance for health policy based on scope note information about subject categories and journal popularity based on impact factors. RESULTS: We found that the majority of Canadian health ministries did not have subscription access to key journals and relied heavily on interlibrary loans. Overall, based on a sample of high-ranking scientific journals, availability of journals through interlibrary loans, online and print-only subscriptions was estimated at 63%, 28% and 3%, respectively. Health Canada had a 2.3-fold higher number of journal subscriptions than that of the provincial ministries' average. Most of the organisations provided access to numerous discipline-specific and multidisciplinary databases. Many organisations provided access to the library resources described through library partnerships or consortia. No professionally led health library environment was found in four out of fourteen Canadian health ministries (i.e. Manitoba Health, Northwest Territories Department of Health and Social Services, Nunavut Department of Health and Social Services and Yukon Department of Health and Social Services). CONCLUSIONS: There is inequity in availability of peer-reviewed research in the fourteen Canadian health ministries. This inequity could present a problem, as each province and territory is responsible for formulating and implementing evidence-informed health policies and services for the benefit of its population.
Asunto(s)
Bases de Datos Bibliográficas/provisión & distribución , Bibliotecas Médicas/provisión & distribución , Servicios de Biblioteca/provisión & distribución , Publicaciones Periódicas como Asunto/provisión & distribución , Canadá , Estudios Transversales , Préstamos entre Bibliotecas/estadística & datos numéricosRESUMEN
We found in the environmental strain Nitrosomonas europaea a chromosomal integron-like structure with an integrase gene, intI(Neu). We have tested the capacity of the IntINeu integrase to excise and integrate several resistance gene cassettes. The results allow us to consider IntINeu a new functional integron integrase.