Dissemination of carbapenem resistance and plasmids encoding carbapenemases in Gram-negative bacteria isolated in India

Acknowledgements We thank Vellore Institute of Technology (VIT) for providing partial funding as ‘VIT Seed Grant’ and Council of Scientific and Industrial Research (CSIR) for providing financial assistance to P.M. in the form of senior research fellowship (SRF) to support this research work. A preprint of this article has been published in BioRxiv and Research Square (Manohar et al.). Funding This study was supported by internal fundingPeer reviewedPublisher PD

Bilayer networks within a hydrogel shell: A robust chassis for artificial cells and a platform for membrane studies

The ability to make artificial lipid bilayers compatible with a wide range of environments, and with sufficient structural rigidity for manual handling, would open up a wealth of opportunities for their more routine use in realworld applications. Although droplet interface bilayers (DIBs) have been demonstrated in a host of laboratory applications, from chemical logic to biosynthesis reaction vessels, their wider use is hampered by a lack of mechanical stability and the largely manual methods employed in their production. Multiphase microfluidics has enabled us to construct hierarchical triple emulsions with a semipermeable shell, in order to form robust, bilayer-bound, droplet networks capable of communication with their external surroundings. These constructs are stable in air, water, and oil environments and overcome a critical obstacle of achieving structural rigidity without compromising environmental interaction. This paves the way for practical application of artificial membranes or droplet networks in diverse areas such as medical applications, drug testing, biophysical studies and their use as synthetic cells

Complete genome sequence of Pseudomonas Phage Motto

We describe the complete genome sequence of bacteriophage Motto, which infects clinical strains of Pseudomonas aeruginosa . Motto is a T1-like siphovirus related to members of the family Drexlerviridae and has a capsid width of ~57 nm and a tail length of ~255 nm. The 49.9-kb genome contains 84 protein-coding genes

In vitro and in vivo evaluation of the biofilm-degrading Pseudomonas phage Motto, as a candidate for phage therapy

Infections caused by Pseudomonas aeruginosa are becoming increasingly difficult to treat due to the emergence of strains that have acquired multidrug resistance. Therefore, phage therapy has gained attention as an alternative to the treatment of pseudomonal infections. Phages are not only bactericidal but occasionally show activity against biofilm as well. In this study, we describe the Pseudomonas phage Motto, a T1-like phage that can clear P. aeruginosa infections in an animal model and also exhibits biofilm-degrading properties. The phage has a substantial anti-biofilm activity against strong biofilm-producing isolates (n = 10), with at least a twofold reduction within 24 h. To demonstrate the safety of using phage Motto, cytotoxicity studies were conducted with human cell lines (HEK 293 and RAW 264.7 macrophages). Using a previously established in vivo model, we demonstrated the efficacy of Motto in Caenorhabditis elegans, with a 90% survival rate when treated with the phage at a multiplicity of infection of 10

Single molecule fluorescence for membrane proteins

The cell membrane is a complex milieu of lipids and proteins. In order to understand the behaviour of individual molecules is it often desirable to examine them as purified components in in vitro systems. Here, we detail the creation and use of droplet interface bilayers (DIBs) which, when coupled to TIRF microscopy, can reveal spatiotemporal and kinetic information for individual membrane proteins. A number of steps are required including modification of the protein sequence to enable the incorporation of appropriate fluorescent labels, expression and purification of the membrane protein and subsequent labelling. Following creation of DIBs, proteins are spontaneously incorporated into the membrane where they can be imaged via conventional single molecule TIRF approaches. Using this strategy, in conjunction with step-wise photobleaching, FRET and / or single particle tracking, a host of parameters can be determined such as oligomerisation state and dynamic information. We discuss advantages and limitations of this system and offer guidance for successful implementation of these approaches

Lignin-based polyurethane materials

Four technical lignins (Alcell, Indulin AT, Sarkanda and Curan 27-11P) were used as macromonomers in the synthesis of polyurethane materials following two global approaches. In the first one Alcell and Indulin AT lignins were used directly as co-monomers in combination with a linear polycaprolactone (PCL) in order to produce polyurethane elastomers where lignin content varied between 10 and 25% (w/w) with respect to polyol mixture (PCL+lignin). The thermomechanical properties of the resulting materials were determined by dynamical mechanical analysis (DMA), differential scanning calorimetry (DSC) and swelling tests. In lignin-based elastomers Indulin AT showed to be more efficiently incorporated in the polyurethane network compared with Alcell lignin. Elastomers prepared with Indulin AT lignin exhibited a cross-linking density and storage modulus (rubbery plateau) higher than those of Alcell lignin-based counterpart and a lower soluble fraction. For both Alcell and Indulin AT based elastomers the glass transition temperature increased and extended over a wide temperature range with the increase of lignin content. The second approach consisted of producing rigid polyurethane foams (RPU) using ligninbased polyols obtained after chemical modification by an oxypropylation procedure. Two polyol formulations (20/80 and 30/70, in what concerns the weight ratios between lignin and propylene oxide, PO), were used in RPU formulations and their content varied from 0 to 100% (w/w with respect to a commercial polyol, used as a reference). The resulting RPU foams were characterized in terms of density, mechanical properties, conductivity and morphology. The prepared RPU foams with lignin-based polyols presented properties, very similar to those obtained from conventional commercial polyols. RPU foams prepared with 30/70 polyols exhibited improved properties comparatively to those arising from 20/80 formulations. Exceptions were however detected in RPU foams prepared with all Sarkanda lignin based polyols and Curan 27-11P 30/70 formulation, which were found to be inadequate for RPU formulation

Interdroplet bilayer arrays in millifluidic droplet traps from 3D-printed moulds

In droplet microfluidics, aqueous droplets are typically separated by an oil phase to ensure containment of molecules in individual droplets of nano-to-picoliter volume. An interesting variation of this method involves bringing two phospholipid-coated droplets into contact to form a lipid bilayer in-between the droplets. These interdroplet bilayers, created by manual pipetting of microliter droplets, have proved advantageous for the study of membrane transport phenomena, including ion channel electrophysiology. In this study, we adapted the droplet microfluidics methodology to achieve automated formation of interdroplet lipid bilayer arrays. We developed a ‘millifluidic’ chip for microliter droplet generation and droplet packing, which is cast from a 3D-printed mould. Droplets of 0.7–6.0 μL volume were packed as homogeneous or heterogeneous linear arrays of 2–9 droplets that were stable for at least six hours. The interdroplet bilayers had an area of up to 0.56 mm2, or an equivalent diameter of up to 850 μm, as determined from capacitance measurements. We observed osmotic water transfer over the bilayers as well as sequential bilayer lysis by the pore-forming toxin melittin. These millifluidic interdroplet bilayer arrays combine the ease of electrical and optical access of manually pipetted microdroplets with the automation and reproducibility of microfluidic technologies. Moreover, the 3D-printing based fabrication strategy enables the rapid implementation of alternative channel geometries, e.g. branched arrays, with a design-to-device time of just 24–48 hours

Complete genome sequence of the virulent Klebsiella pneumoniae Phage Geezett infecting multidrug-resistant clinical strains

Geezett was isolated from hospital sewage in Hangzhou, China, and exhibits lytic activity against clinical isolates of the nosocomial pathogen Klebsiella pneumoniae. The bacteriophage is a myovirus and has a double-stranded DNA (dsDNA) genome 50,707 bp long, containing 79 open reading frames (ORFs)

Co‐evolutionary adaptations of Acinetobacter baumannii and a clinical carbapenemase‐encoding plasmid during carbapenem exposure

Abstract: OXA‐23 is the predominant carbapenemase in carbapenem‐resistant Acinetobacter baumannii. The co‐evolutionary dynamics of A. baumannii and OXA‐23‐encoding plasmids are poorly understood. Here, we transformed A. baumannii ATCC 17978 with pAZJ221, a blaOXA−23‐containing plasmid from clinical A. baumannii isolate A221, and subjected the transformant to experimental evolution in the presence of a sub‐inhibitory concentration of imipenem for nearly 400 generations. We used population sequencing to track genetic changes at six time points and evaluated phenotypic changes. Increased fitness of evolving populations, temporary duplication of blaOXA−23 in pAZJ221, interfering allele dynamics, and chromosomal locus‐level parallelism were observed. To characterize genotype‐to‐phenotype associations, we focused on six mutations in parallel targets predicted to affect small RNAs and a cyclic dimeric (3′ → 5′) GMP‐metabolizing protein. Six isogenic mutants with or without pAZJ221 were engineered to test for the effects of these mutations on fitness costs and plasmid kinetics, and the evolved plasmid containing two copies of blaOXA−23 was transferred to ancestral ATCC 17978. Five of the six mutations contributed to improved fitness in the presence of pAZJ221 under imipenem pressure, and all but one of them impaired plasmid conjugation ability. The duplication of blaOXA−23 increased host fitness under carbapenem pressure but imposed a burden on the host in antibiotic‐free media relative to the ancestral pAZJ221. Overall, our study provides a framework for the co‐evolution of A. baumannii and a clinical blaOXA−23‐containing plasmid in the presence of imipenem, involving early blaOXA−23 duplication followed by chromosomal adaptations that improved the fitness of plasmid‐carrying cells