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

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

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

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

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

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

The Transmembrane Morphogenesis Protein gp1 of Filamentous Phages Contains Walker A and Walker B Motifs Essential for Phage Assembly

In contrast to lytic phages, filamentous phages are assembled in the inner membrane and secreted across the bacterial envelope without killing the host. For assembly and extrusion of the phage across the host cell wall, filamentous phages code for membrane-embedded morphogenesis proteins. In the outer membrane of Escherichia coli, the protein gp4 forms a pore-like structure, while gp1 and gp11 form a complex in the inner membrane of the host. By comparing sequences with other filamentous phages, we identified putative Walker A and B motifs in gp1 with a conserved lysine in the Walker A motif (K14), and a glutamic and aspartic acid in the Walker B motif (D88, E89). In this work we demonstrate that both, Walker A and Walker B, are essential for phage production. The crucial role of these key residues suggests that gp1 might be a molecular motor driving phage assembly. We further identified essential residues for the function of the assembly complex. Mutations in three out of six cysteine residues abolish phage production. Similarly, two out of six conserved glycine residues are crucial for gp1 function. We hypothesise that the residues represent molecular hinges allowing domain movement for nucleotide binding and phage assembly