The cost-effectiveness of a patient centred pressure ulcer prevention care bundle: findings from the INTACT cluster randomised trial

Background: Pressure ulcers are serious, avoidable, costly and common adverse outcomes of healthcare. Objectives: To evaluate the cost-effectiveness of a patient-centred pressure ulcer prevention care bundle compared to standard care. Design: Cost-effectiveness and cost-benefit analyses of pressure ulcer prevention performed from the health system perspective using data collected alongside a cluster-randomised trial. Settings: Eight tertiary hospitals in Australia. Participants: Adult patients receiving either a patient-centred pressure ulcer prevention care bundle (n = 799) or standard care (n = 799). Methods: Direct costs related to the intervention and preventative strategies were collected from trial data and supplemented by micro-costing data on patient turning and skin care from a 4-week substudy (n = 317). The time horizon for the economic evaluation matched the trial duration, with the endpoint being diagnosis of a new pressure ulcer, hospital discharge/transfer or 28 days; whichever occurred first. For the cost-effectiveness analysis, the primary outcome was the incremental costs of prevention per additional hospital acquired pressure ulcer case avoided, estimated using a two-stage cluster-adjusted non-parametric bootstrap method. The cost-benefit analysis estimated net monetary benefit, which considered both the costs of prevention and any difference in length of stay. All costs are reported in AU$(2015). Results: The care bundle cost AU$144.91 (95%CI: $74.96 to$246.08) more per patient than standard care. The largest contributors to cost were clinical nurse time for repositioning and skin inspection. In the cost-effectiveness analysis, the care bundle was estimated to cost an additional $3,296 (95$144,525) per pressure ulcer avoided. This estimate is highly uncertain. Length of stay was unexpectedly higher in the care bundle group. In a cost-benefit analysis which considered length of stay, the net monetary benefit for the care bundle was estimated to be −$2,320 (95$3,900, −$1,175) per patient, suggesting the care bundle was not a cost-effective use of resources. Conclusions: A pressure ulcer prevention care bundle consisting of multicomponent nurse training and patient education may promote best practice nursing care but may not be cost-effective in preventing hospital acquired pressure ulcer

Inverse Correlation between Promoter Strength and Excision Activity in Class 1 Integrons

Class 1 integrons are widespread genetic elements that allow bacteria to capture and express gene cassettes that are usually promoterless. These integrons play a major role in the dissemination of antibiotic resistance among Gram-negative bacteria. They typically consist of a gene (intI) encoding an integrase (that catalyzes the gene cassette movement by site-specific recombination), a recombination site (attI1), and a promoter (Pc) responsible for the expression of inserted gene cassettes. The Pc promoter can occasionally be combined with a second promoter designated P2, and several Pc variants with different strengths have been described, although their relative distribution is not known. The Pc promoter in class 1 integrons is located within the intI1 coding sequence. The Pc polymorphism affects the amino acid sequence of IntI1 and the effect of this feature on the integrase recombination activity has not previously been investigated. We therefore conducted an extensive in silico study of class 1 integron sequences in order to assess the distribution of Pc variants. We also measured these promoters' strength by means of transcriptional reporter gene fusion experiments and estimated the excision and integration activities of the different IntI1 variants. We found that there are currently 13 Pc variants, leading to 10 IntI1 variants, that have a highly uneven distribution. There are five main Pc-P2 combinations, corresponding to five promoter strengths, and three main integrases displaying similar integration activity but very different excision efficiency. Promoter strength correlates with integrase excision activity: the weaker the promoter, the stronger the integrase. The tight relationship between the aptitude of class 1 integrons to recombine cassettes and express gene cassettes may be a key to understanding the short-term evolution of integrons. Dissemination of integron-driven drug resistance is therefore more complex than previously thought

In Vitro Recombination Catalyzed by Bacterial Class 1 Integron Integrase IntI1 Involves Cooperative Binding and Specific Oligomeric Intermediates

Gene transfer via bacterial integrons is a major pathway for facilitating the spread of antibiotic resistance genes across bacteria. Recently the mechanism underlying the recombination catalyzed by class 1 integron recombinase (IntI1) between attC and attI1 was highlighted demonstrating the involvement of a single-stranded intermediary on the attC site. However, the process allowing the generation of this single-stranded substrate has not been determined, nor have the active IntI1•DNA complexes been identified. Using the in vitro strand transfer assay and a crosslink strategy we previously described we demonstrated that the single-stranded attC sequences could be generated in the absence of other bacterial proteins in addition to IntI. This suggests a possible role for this protein in stabilizing and/or generating this structure. The mechanism of folding of the active IntI•DNA complexes was further analyzed and we show here that it involves a cooperative binding of the protein to each recombination site and the emergence of different oligomeric species specific for each DNA substrate. These findings provide further insight into the recombination reaction catalyzed by IntI1

DNA Adenine Methylation Is Required to Replicate Both Vibrio cholerae Chromosomes Once per Cell Cycle

DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated) sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII) of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication

Chloramphenicol Selection of IS10 Transposition in the cat Promoter Region of Widely Used Cloning Vectors

The widely used pSU8 family of cloning vectors is based on a p15A replicon and a chloramphenicol acetyltransferase (cat) gene conferring chloramphenicol resistance. We frequently observed an increase in the size of plasmids derived from these vectors. Analysis of the bigger molecular species shows that they have an IS10 copy inserted at a specific site between the promoter and the cat open reading frame. Promoter activity from both ends of IS10 has been reported, suggesting that the insertion events could lead to higher CAT production. Insertions were observed in certain constructions containing inserts that could lead to plasmid instability. To test the possibility that IS10 insertions were selected as a response to chloramphenicol selection, we have grown these constructs in the presence of different amounts of antibiotic and we observed that insertions arise promptly under higher chloramphenicol selective pressure. IS10 is present in many E. coli laboratory strains, so the possibility of insertion in constructions involving cat-containing vectors should be taken into account. Using lower chloramphenicol concentrations could solve this problem

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. [Methodology/Principal Findings]: We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. [Conclusions/Significance]: The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.This work was supported by grant BIO2008-00133 from the Spanish Ministry of Science and Innovation to ML. CGP was a recipient of a predoctoral fellowship from the University of Cantabria, Spain.Peer reviewe

Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome

<p><u>(A) Circular map of the <i>E</i>. <i>coli</i> chromosome</u>: <i>oriC</i>, <i>dif</i> and <i>terD</i> to <i>terB</i> sites are indicated. Numbers refer to the chromosome coordinates (in kb) of MG1655. (<u>B) Linear map of the terminus region:</u> chromosome coordinates are shown increasing from left to right, as in the marker frequency panels (see Figure 1C for example), therefore in the opposite direction to the circular map. In addition to <i>dif</i> and <i>ter</i> sites, the positions of the <i>parS</i>_pMT1 sites used for microscopy experiments are indicated. (<u>C) MFA analysis of terminus DNA loss in the <i>recB</i> mutant</u>: sequence read frequencies of exponential phase cells normalized to the total number of reads were calculated for each strain. Ratios of normalized reads in isogenic wild-type and <i>recB</i> mutant are plotted against chromosomal coordinates (in kb). The profile ratio of the terminus region is enlarged and the profile of the corresponding entire chromosomes is shown in inset. Original normalized profiles used to calculate ratios are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007256#pgen.1007256.s005" target="_blank">S1 Fig</a>. The position of <i>dif</i> is indicated by a red arrow. The <i>ter</i> sites that arrest clockwise forks (<i>terC</i>, <i>terB</i>, green arrow) and counter-clockwise forks (<i>terA</i>, <i>terD</i>, blue arrow) are shown. <u>(D) Schematic representation of focus loss in the <i>recB</i> mutant:</u> Time-lapse microscopy experiments showed that loss of a focus in the <i>recB</i> mutant occurs concomitantly with cell division in one of two daughter cells, and that the cell that keeps the focus then generates a focus-less cell at each generation. The percentage of initial events was calculated as the percentage of cell divisions that generate a focus-less cell, not counting the following generations. In this schematic representation, two initial events occurred (generations #2 and #7) out of 9 generations, and focus loss at generation #2 is heritable. Panels shown in this figure were previously published in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007256#pgen.1007256.ref019" target="_blank">19</a>] and are reproduced here to introduce the phenomenon.</p

The emergence of Vibrio pathogens in Europe: ecology, evolution, and pathogenesis

Global change has caused a worldwide increase in reports of Vibrio-associated diseases with ecosystem-wide impacts on humans and marine animals. In Europe, higher prevalence of human infections followed regional climatic trends with outbreaks occurring during episodes of unusually warm weather. Similar patterns were also observed in Vibrio-associated diseases affecting marine organisms such as fish, bivalves and corals. Basic knowledge is still lacking on the ecology and evolutionary biology of these bacteria as well as on their virulence mechanisms. Current limitations in experimental systems to study infection and the lack of diagnostic tools still prevent a better understanding of Vibrio emergence. A major challenge is to foster cooperation between fundamental and applied research in order to investigate the consequences of pathogen emergence in natural Vibrio populations and answer federative questions that meet societal needs. Here we report the proceedings of the first European workshop dedicated to these specific goals of the Vibrio research community by connecting current knowledge to societal issues related to ocean health and food security

Regulatory Cross-Talk Links Vibrio cholerae Chromosome II Replication and Segregation

There is little knowledge of factors and mechanisms for coordinating bacterial chromosome replication and segregation. Previous studies have revealed that genes (and their products) that surround the origin of replication (oriCII) of Vibrio cholerae chromosome II (chrII) are critical for controlling the replication and segregation of this chromosome. rctB, which flanks one side of oriCII, encodes a protein that initiates chrII replication; rctA, which flanks the other side of oriCII, inhibits rctB activity. The chrII parAB2 operon, which is essential for chrII partitioning, is located immediately downstream of rctA. Here, we explored how rctA exerts negative control over chrII replication. Our observations suggest that RctB has at least two DNA binding domains—one for binding to oriCII and initiating replication and the other for binding to rctA and thereby inhibiting RctB's ability to initiate replication. Notably, the inhibitory effect of rctA could be alleviated by binding of ParB2 to a centromere-like parS site within rctA. Furthermore, by binding to rctA, ParB2 and RctB inversely regulate expression of the parAB2 genes. Together, our findings suggest that fluctuations in binding of the partitioning protein ParB2 and the chrII initiator RctB to rctA underlie a regulatory network controlling both oriCII firing and the production of the essential chrII partitioning proteins. Thus, by binding both RctB and ParB2, rctA serves as a nexus for regulatory cross-talk coordinating chrII replication and segregation

Evidence for the Role of Horizontal Transfer in Generating pVT1, a Large Mosaic Conjugative Plasmid from the Clam Pathogen, Vibrio tapetis

The marine bacterium Vibrio tapetis is the causative agent of the brown ring disease, which affects the clam Ruditapes philippinarum and causes heavy economic losses in North of Europe and in Eastern Asia. Further characterization of V. tapetis isolates showed that all the investigated strains harbored at least one large plasmid. We determined the sequence of the 82,266 bp plasmid pVT1 from the CECT4600T reference strain and analyzed its genetic content. pVT1 is a mosaic plasmid closely related to several conjugative plasmids isolated from Vibrio vulnificus strains and was shown to be itself conjugative in Vibrios. In addition, it contains DNA regions that have similarity with several other plasmids from marine bacteria (Vibrio sp., Shewanella sp., Listonella anguillarum and Photobacterium profundum). pVT1 contains a number of mobile elements, including twelve Insertion Sequences or inactivated IS genes and an RS1 phage element related to the CTXphi phage of V. cholerae. The genetic organization of pVT1 underscores an important role of horizontal gene transfer through conjugative plasmid shuffling and transposition events in the acquisition of new genetic resources and in generating the pVT1 modular organization. In addition, pVT1 presents a copy number of 9, relatively high for a conjugative plasmid, and appears to belong to a new type of replicon, which may be specific to Vibrionaceae and Shewanelleacae