Novel bacteriophage therapy for controlling metallo-beta-lactamase producing Pseudomonas aeruginosa infection in Catfish

Background: The bacteriophage therapy is an effective antimicrobial approach with potentially important applications in medicine and biotechnology which can be seen as an additional string in the bow. Emerging drug resistant bacteria in aquaculture industry due to unrestricted use of antibiotics warrants more sustainable and environmental friendly strategies for controlling fish infections. The isolated bacteria from fish lesions was characterised based on isolation on selective and differential medium like Pseudomonas agar, gram staining, biochemical tests and 16SrRNA sequencing. The metallo-beta-lactamase (MBL) producing bacterial isolate was evaluated using Imipenem - Ethylenediaminetetraacetic acid (EDTA) disk method. The specific bacteriophage was isolated and concentrated using coal bed developed in our lab at CSIR-NEERI. The isolated and enriched bacteriophage was characterised by nucleotide sequencing and electron microscopy. The phage therapy was applied for treating ulcerative lesion in fish. Results: The pathogenic bacterium responsible for causing ulcerative lesions in catfish species (Clarias gariepinus) was identified as Pseudomonas aeruginosa. One out of twenty P. aeruginosa isolate showing multi drug resistance (MDR) was incidentally found to be MBL producing as determined by Imipenem-EDTA disk method. The phage therapy effectively cured the ulcerative lesions of the infected fish in 8–10 days of treatment, with a sevenfold reduction of the lesion with untreated infection control. Conclusion: Bacteriophage therapy can have potential applications soon as an alternative or as a complement to antibiotic treatment in the aquaculture. We present bacteriophage therapy as a treatment method for controlling MDR P. aeruginosa infection in C. gariepinus. To the best of our knowledge this is a first report of application of phage therapy against MBL producing P. aeruginosa isolated from aquatic ecosystem. Keywords: P. aeruginosa, Multi drug resistance, Metallo-β-lactamase, Bacteriophage therapy, Catfis

Alcoholic fermentation of thermochemical and biological hydrolysates derived from Miscanthus biomass by Clostridium acetobutylicum ATCC 824

© 2019 This laboratory scale study aims to demonstrate the effectiveness of thermochemical and biological saccharification of Miscanthus giganteus (MG) for generation of fermentable saccharides and its subsequent fermentation into solvents i.e. acetone, ethanol and butanol (ABE) using Clostridium acetobutylicum ATCC 824. Saccharide hydrolysates were derived from MG by thermochemical (water, acid and alkali at 130 °C) and biological saccharification (Fibrobacter succinogenes S85) processes and were subjected to batch fermentation for 120 h using C. acetobutylicum ATCC 824. At the end of fermentation of thermochemically-derived hydrolysates, 742 g m−3 of saccharides from water treatment, 9572 g m−3 of saccharides from acid treatment and 4054 g m−3 of saccharides from alkali treatment were fermented and yielded 0.045, 0.0069 and 0.01 g g−1 of total solvents, respectively. Similarly, at the end of fermentation of biological hydrolysate (using F. succinogenes), 2504 g m−3 of saccharides was fermented and yielded 0.091 g g−1 of total solvents. The highest yield of total solvents was achieved by water (thermochemical) and biological saccharification of MG using C. acetobutylicum. Whereas, acid and alkali-treated hydrolysates showed lower yields of solvents presumably due to production of inhibitory compounds during saccharification. Compared to thermochemical saccharification, biological saccharification using F. succinogenes is a promising approach since it yielded the highest amount of solvents whilst being eco-friendly. Our future studies will focus on optimisation of biological saccharification (using F. succinogenes) and sequential co-culture fermentation (using C. acetobutylicum). The development of alternative consolidated bioprocessing approach using biological saccharification will contribute towards making lignocellulosic biofuels a reality

Influence of Substrates on the Surface Characteristics and Membrane Proteome of Fibrobacter succinogenes S85

Although Fibrobacter succinogenes S85 is one of the most proficient cellulose degrading bacteria among all mesophilic organisms in the rumen of herbivores, the molecular mechanism behind cellulose degradation by this bacterium is not fully elucidated. Previous studies have indicated that cell surface proteins might play a role in adhesion to and subsequent degradation of cellulose in this bacterium. It has also been suggested that cellulose degradation machinery on the surface may be selectively expressed in response to the presence of cellulose. Based on the genome sequence, several models of cellulose degradation have been suggested. The aim of this study is to evaluate the role of the cell envelope proteins in adhesion to cellulose and to gain a better understanding of the subsequent cellulose degradation mechanism in this bacterium. Comparative analysis of the surface (exposed outer membrane) chemistry of the cells grown in glucose, acid-swollen cellulose and microcrystalline cellulose using physico-chemical characterisation techniques such as electrophoretic mobility analysis, microbial adhesion to hydrocarbons assay and Fourier transform infra-red spectroscopy, suggest that adhesion to cellulose is a consequence of an increase in protein display and a concomitant reduction in the cell surface polysaccharides in the presence of cellulose. In order to gain further understanding of the molecular mechanism of cellulose degradation in this bacterium, the cell envelope-associated proteins were enriched using affinity purification and identified by tandem mass spectrometry. In total, 185 cell envelope-associated proteins were confidently identified. Of these, 25 proteins are predicted to be involved in cellulose adhesion and degradation, and 43 proteins are involved in solute transport and energy generation. Our results supports the model that cellulose degradation in F. succinogenes occurs at the outer membrane with active transport of cellodextrins across for further metabolism of cellodextrins to glucose in the periplasmic space and inner cytoplasmic membrane

Quantitative proteomic analysis of the influence of lignin on biofuel production by Clostridium acetobutylicum ATCC 824

Background: Clostridium acetobutylicum has been a focus of research because of its ability to produce high-value compounds that can be used as biofuels. Lignocellulose is a promising feedstock, but the lignin–cellulose–hemicellulose biomass complex requires chemical pre-treatment to yield fermentable saccharides, including cellulose-derived cellobiose, prior to bioproduction of acetone–butanol–ethanol (ABE) and hydrogen. Fermentation capability is limited by lignin and thus process optimization requires knowledge of lignin inhibition. The effects of lignin on cellular metabolism were evaluated for C. acetobutylicum grown on medium containing either cellobiose only or cellobiose plus lignin. Microscopy, gas chromatography and 8-plex iTRAQ-based quantitative proteomic technologies were applied to interrogate the effect of lignin on cellular morphology, fermentation and the proteome. Results: Our results demonstrate that C. acetobutylicum has reduced performance for solvent production when lignin is present in the medium. Medium supplemented with 1 g L−1 of lignin led to delay and decreased solvents production (ethanol; 0.47 g L−1 for cellobiose and 0.27 g L−1 for cellobiose plus lignin and butanol; 0.13 g L−1 for cellobiose and 0.04 g L−1 for cellobiose plus lignin) at 20 and 48 h, respectively, resulting in the accumulation of acetic acid and butyric acid. Of 583 identified proteins (FDR < 1 %), 328 proteins were quantified with at least two unique peptides. Up- or down-regulation of protein expression was determined by comparison of exponential and stationary phases of cellobiose in the presence and absence of lignin. Of relevance, glycolysis and fermentative pathways were mostly down-regulated, during exponential and stationary growth phases in presence of lignin. Moreover, proteins involved in DNA repair, transcription/translation and GTP/ATP-dependent activities were also significantly affected and these changes were associated with altered cell morphology. Conclusions: This is the first comprehensive analysis of the cellular responses of C. acetobutylicum to lignin at metabolic and physiological levels. These data will enable targeted metabolic engineering strategies to optimize biofuel production from biomass by overcoming limitations imposed by the presence of lignin

Bacterial Cellulose-Based Blends and Composites: Versatile Biomaterials for Tissue Engineering Applications

Cellulose of bacterial origin, known as bacterial cellulose (BC), is one of the most versatile biomaterials that has a huge potential in tissue engineering due to its favourable mechanical properties, high hydrophilicity, crystallinity, and purity. Additional properties such as porous nano-fibrillar 3D structure and a high degree of polymerisation of BC mimic the properties of the native extracellular matrix (ECM), making it an excellent material for the fabrication of composite scaffolds suitable for cell growth and tissue development. Recently, the fabrication of BC-based scaffolds, including composites and blends with nanomaterials, and other biocompatible polymers has received particular attention owing to their desirable properties for tissue engineering. These have proven to be promising advanced materials in hard and soft tissue engineering. This review presents the latest state-of-the-art modified/functionalised BC-based composites and blends as advanced materials in tissue engineering. Their applicability as an ideal biomaterial in targeted tissue repair including bone, cartilage, vascular, skin, nerve, and cardiac tissue has been discussed. Additionally, this review briefly summarises the latest updates on the production strategies and characterisation of BC and its composites and blends. Finally, the challenges in the future development and the direction of future research are also discussed

Bacterial Cellulose-Based Blends and Composites: Versatile Biomaterials for Tissue Engineering Applications

Cellulose of bacterial origin, known as bacterial cellulose (BC), is one of the most versatile biomaterials that has a huge potential in tissue engineering due to its favourable mechanical properties, high hydrophilicity, crystallinity, and purity. Additional properties such as porous nano-fibrillar 3D structure and a high degree of polymerisation of BC mimic the properties of the native extracellular matrix (ECM), making it an excellent material for the fabrication of composite scaffolds suitable for cell growth and tissue development. Recently, the fabrication of BC-based scaffolds, including composites and blends with nanomaterials, and other biocompatible polymers has received particular attention owing to their desirable properties for tissue engineering. These have proven to be promising advanced materials in hard and soft tissue engineering. This review presents the latest state-of-the-art modified/functionalised BC-based composites and blends as advanced materials in tissue engineering. Their applicability as an ideal biomaterial in targeted tissue repair including bone, cartilage, vascular, skin, nerve, and cardiac tissue has been discussed. Additionally, this review briefly summarises the latest updates on the production strategies and characterisation of BC and its composites and blends. Finally, the challenges in the future development and the direction of future research are also discussed

Principal component analysis (PCA) of ATR-FTIR spectra of <i>F</i>. <i>succinogenes</i> S85 cells grown on (●) Glucose, (▲) AS cellulose, (♦) MC cellulose.

<p>Principal component analysis (PCA) of ATR-FTIR spectra of <i>F</i>. <i>succinogenes</i> S85 cells grown on (●) Glucose, (▲) AS cellulose, (♦) MC cellulose.</p

Electrophoretic mobility of <i>F</i>. <i>succinogenes</i> S85 cells under different carbon substrate conditions as a function of pH.

<p>Error bars = SE value.</p

List of predicted cell envelope proteins associated with cellulose degradation in <i>F</i>. <i>succinogenes</i> S85.

<p>* G–glucose; MC–microcrystalline cellulose; AS–acid swollen cellulose. The numbers under these columns represent the number of unique valid peptide sequences on which protein identification is based.</p><p>^aCarbohydrate active enzymes database (<a href="http://www.cazy.org/" target="_blank">http://www.cazy.org/</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141197#pone.0141197.ref040" target="_blank">40</a>]</p><p>^bLocation of the given proteins predicted by the PSORTb subcellular localization prediction tool version 3.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141197#pone.0141197.ref037" target="_blank">37</a>]</p><p>^cTheoretical isoelectric point, molecular mass and gravy index of the given protein, as predicted by the ExPASy Compute pI/MW tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141197#pone.0141197.ref038" target="_blank">38</a>]</p><p>^dDetermined by SignalP v.3.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141197#pone.0141197.ref039" target="_blank">39</a>] the numbers in parentheses indicates the amino acids between which cleavage is predicted to occur in the given protein</p><p>List of predicted cell envelope proteins associated with cellulose degradation in <i>F</i>. <i>succinogenes</i> S85.</p

Transmission electron microscopy (TEM) images of the bacterium <i>F</i>. <i>succinogenes</i> S85 cells grown on glucose (A) and cellulose substrate (B).

<p>Transmission electron microscopy (TEM) images of the bacterium <i>F</i>. <i>succinogenes</i> S85 cells grown on glucose (A) and cellulose substrate (B).</p