Impact of daily octenidine skin washing versus nonwashing on antiseptic tolerance of coagulase-negative staphylococci in two neonatal intensive care units with different skin cleansing practices.

Background: There is wide variation in practices regarding routine bathing/washing of babies in neonatal intensive care units (NICUs). Evidence is lacking as to the benefit of routine antiseptic washes for reducing infection. We aimed to compare the antiseptic tolerance of Coagulase Negative Staphylococci (CoNS) within two UK NICUs with very different approaches to skin washing. Methods: We compared antiseptic susceptibility of CoNS isolated from skin swabs of neonates admitted to the Norfolk and Norwich University Hospital (NNUH) NICU in December 2017-March 2018 with those isolated in the Bradford Royal Infirmary (BRI) NICU in January-March 2020. The NNUH does not practise routine whole-body washing whereas BRI practises daily whole-body washing from post-menstrual age 27 weeks using Octenisan wash lotion (0.3% octenidine; 1 minute contact time before washing off with sterile water). A total of 78 CoNS isolates from BRI and 863 from the NNUH were tested for susceptibility against the antiseptics octenidine (OCT) and chlorhexidine (CHX). Results: Isolates from the BRI with practice of routine washing did not show increased antiseptic tolerance to OCT or CHX. Isolates from the NNUH which does not practise routine whole-body washing and rarely uses octenidine, were comparatively less susceptible to both CHX and OCT antiseptics. Conclusions: Daily whole-body skin washing with OCT does not appear to select for CoNS isolates that are antiseptic tolerant towards OCT and CHX. There remains considerable uncertainty about the impact of different antiseptic regimes on neonatal skin microbiota, the benefit of routine washing, and the development of antiseptic tolerance in the NICU.

CRISPR-mediated knock-in of transgenes into the malaria vector Anopheles funestus.

The ability to introduce mutations, or transgenes, of choice to precise genomic locations has revolutionized our ability to understand how genes and organisms work. In many mosquito species that are vectors of various human diseases, the advent of CRISPR genome editing tools has shed light on basic aspects of their biology that are relevant to their efficiency as disease vectors. This allows a better understanding of how current control tools work and opens up the possibility of novel genetic control approaches, such as gene drives, that deliberately introduce genetic traits into populations. Yet for the Anopheles funestus mosquito, a significant vector of malaria in sub-Saharan Africa and indeed the dominant vector species in many countries, transgenesis has yet to be achieved. We describe herein an optimized transformation system based on the germline delivery of CRISPR components that allows efficient cleavage of a previously validated genomic site and preferential repair of these cut sites via homology-directed repair (HDR), which allows the introduction of exogenous template sequence, rather than end-joining repair. The rates of transformation achieved are sufficiently high that it should be able to introduce alleles of choice to a target locus, and recover these, without the need to include additional dominant marker genes. Moreover, the high rates of HDR observed suggest that gene drives, which employ an HDR-type mechanism to ensure their proliferation in the genome, may be well suited to work in A. funestus.

Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics.

CRISPR guide RNAs (gRNAs) can be programmed with relative ease to allow the genetic editing of nearly any DNA or RNA sequence. Here, we propose novel molecular architectures to achieve RNA-dependent modulation of CRISPR activity in response to specific RNA molecules. We designed and tested, in both living Escherichia coli cells and cell-free assays for rapid prototyping, cis-repressed RNA-interacting guide RNA (igRNA) that switch to their active state only upon interaction with small RNA fragments or long RNA transcripts, including pathogen-derived mRNAs of medical relevance such as the human immunodeficiency virus infectivity factor. The proposed CRISPR-igRNAs are fully customizable and easily adaptable to the majority if not all the available CRISPR-Cas variants to modulate a variety of genetic functions in response to specific cellular conditions, providing orthogonal activation and increased specificity. We thereby foresee a large scope of application for therapeutic, diagnostic, and biotech applications in both prokaryotic and eukaryotic systems.

Nuclease-based gene drives, an innovative tool for insect vector control: advantages and challenges of the technology.

Genetic control of insects involves the release of modified insects that contain altered genetic traits and are competent to mate with target populations to introduce the traits therein. Since it relies on mating, this type of control is species-specific, non-toxic, and has the advantage that the released insects can do the difficult task of reaching remote and otherwise inaccessible insect niches. Gene drives are capable of drastically biasing their own transmission and are being developed as a new type of genetic control, one that would be self-sustaining, requiring low numbers in the initial release in order to spread and persist within a population. In this review, the advantages and challenges of building and deploying this technology will be discussed, using mosquito control as an example.

Overexpression of Fgfr2c causes craniofacial bone hypoplasia and ameliorates craniosynostosis in the Crouzon mouse.

FGFR2c regulates many aspects of craniofacial and skeletal development. Mutations in the FGFR2 gene are causative of multiple forms of syndromic craniosynostosis, including Crouzon syndrome. Paradoxically, mouse studies have shown that the activation (Fgfr2cC342Y; a mouse model for human Crouzon syndrome), as well as the removal (Fgfr2cnull), of the FGFR2c isoform can drive suture abolishment. This study aims to address the downstream effects of pathogenic FGFR2c signalling by studying the effects of Fgfr2c overexpression. Conditional overexpression of Fgfr2c (R26RFgfr2c;ßact) results in craniofacial hypoplasia as well as microtia and cleft palate. Contrary to Fgfr2cnull and Fgfr2cC342Y, Fgfr2c overexpression is insufficient to drive onset of craniosynostosis. Examination of the MAPK/ERK pathway in the embryonic sutures of Fgfr2cC342Y and R26RFgfr2c;ßact mice reveals that both mutants have increased pERK expression. The contrasting phenotypes between Fgfr2cC342Y and R26RFgfr2c;ßact mice prompted us to assess the impact of the Fgfr2c overexpression allele on the Crouzon mouse (Fgfr2cC342Y), in particular its effects on the coronal suture. Our results demonstrate that Fgfr2c overexpression is sufficient to partially rescue craniosynostosis through increased proliferation and reduced osteogenic activity in E18.5 Fgfr2cC342Y embryos. This study demonstrates the intricate balance of FGF signalling required for correct calvarial bone and suture morphogenesis, and that increasing the expression of the wild-type FGFR2c isoform could be a way to prevent or delay craniosynostosis progression.