DOCSLIB.ORG

Gram-Negative Bacteria; for Instance, Coetzee Spheroplast Formation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Gram-Negative Bacteria; for Instance, Coetzee Spheroplast Formation Agric. Biol. Chem., 49 (1), 133-140, 1985 133 Fusion of Spheroplasts and Genetic Recombination of Zymomonasmobilis Hideshi Yanase, Masaru Yasui, Takayuki Miyazaki and KenzoTonomura Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Mozu-ume-machi, Sakai, Osaka 591, Japan Received July 1.8, 1984 Spheroplasts of Zymomonasmobilis (Z-6) were prepared by cultivating it in a hypertonic mediumcontaining penicillin G or glycine at 30°C for more than 6 hr. Thereversion of spheroplasts to a bacillary form was observed at frequencies of 10~2 to 10~3 whenspheroplasts were incubated on hypertonic solid agar plates overlaid with soft agar. Spheroplast fusion was attempted with polyethylene glycol 6000 by crossing two mutants lacking the ability to ferment sugars. Fusants were selected, and obtained at high frequencies. The properties of a stable fusant are discussed. Recently broad host-range plasmids have Media. For the cultivation of Zymomonasstrains, RM and T media were used. RMmediumcontained 2% been transferred into Zymomonasmobilis by glucose, 1.0% yeast extract and 0.2% KH2PO4, pH 6.0. T conjugation.1 ~4) However, transformation medium contained 10% glucose, 1% yeast extract, 1% and transduction have not yet been accom- KH2PO4, 1% (NH4)2SO4 and 0.05% MgSO4-7H2O, pH plished. Besides, cell fusion could be regarded 5.6. For the formation of spheroplasts, S mediumwas used as a useful method for strain improvement. containing 20%sucrose, 2%glucose, 1%yeast extract, There have been manyreports indicating pro- 0.2% KH2PO4 and 0.2% MgSO4à"7H2O, pH 6.0. For the toplast fusion in Gram-positive bacteria such regeneration, R mediumwas used containing 16% sor- as Bacillus^ Staphylococcus,6) Brevibacte- bitol, 2% glucose, 1% yeast extract and 0.2% KH2PO4, pH riurn1) and Streptomyces^ but only a few on Gram-negative bacteria; for instance, Coetzee Spheroplast formation. An aliquot (0.1 ml) of a culture et al.9) have reported intraspecific fusion in of a Zymomonasstrain was inoculated into 5ml of T medium and cultured for 12hr at 30°C. The culture at the Providence alcalifaciens, and Rastogi et al.10) late exponential phase (5 x 108 cells/ml) was diluted 1 : 4 in Mycobacterium aurum. Therefore, we de- 6.0.with S medium containing 3.8% glycine or 250 units/ml of cided to attempt spheroplast fusion in Z. penicillin G. After 6 to 12hr incubation, almost all of the mobilis, and obtained fusants at high frequen- vegetative cells had been converted to a spherical form. cies. This paper describes the procedure for The total numberof cells in the spheroplast mixture was spheroplast formation, spheroplast fusion, and determined with a haemocytometerunder a microscope. cell wall regeneration of Z. mobilis for the first Table I. Strains of ZymomonasUsed time. _ T ^ , Fermentationofstrain Invertase a-Gal _ c oRaf Sue Era MATERIALS AND METHODS Z -6 W In d u c ib le Bacterial strains. These are listed in Table I. Zymomonas Z -6 -C M C o n stitu tiv e - mobilis subsp. mobilis IFO 13756 (Z-6) is a wild strain. The Z - 6 - C f r i T M C o n s t i t u t i v e - Z-6-C strain is a spontaneous mutant derived from Z-6, Z - 6 - C s u e " M C o n s t i t u t i v e - and produces invertase constitutively. Z-6-C fru ~ and Z-6- C sue" are mutants derived from Z-6-C by TV-methyl-JV'- Z-6, Zymomonas mobilis subsp. mobilis IFO13756; nitro-TV-nitrosoguanidine (NTG) treatment, which have Raf, raffinose; Sue, sucrose; Fru, fructose; a-Gal, a- lost the ability to ferment fructose or sucrose, respectively. galactosidase; W, wild; M, mutant. 134 H. Yanase et al. The numbers of osmotic resistant cells in the spheroplast Electron microscopy of spheroplasts. Spheroplasts were mixture were determined as follows. The spheroplast fixed by a modification of the method of Sagara et al.ll) mixture was diluted with 0.85% saline solution to expose This involved treatment with 3%(v/v) glutaraldehyde in the cells to low osmotic pressure, and spread onto an agar phosphate buffer (pH 7.0) containing 1 m sucrose followed plate of RMmedium. The numbers of colonies which by post-fixation in 1 %(w/v) OsO4 for 4hr; both steps were appeared were determined after 5 days incubation at 30°C, performed at 4°C. The fixed material was dehydrated in a and defined as osmotic resistant cells. The number of graded ethanol dilution series and embeddedin Epon 812. spheroplasts in the spheroplast mixture was calculated by This sections were prepared with a LKBultramicrotome deducting the number of osmotic resistant cells from the with glass knives, and viewed under a Hitachi H-300 number of cells. The frequency of spheroplast formation Electron Microscope. wasexpressed as the ratio of the numberof spheroplasts to the total numberof cells. Chemicals. Penicillin G was purchased from Meiji Seika Co., Ltd. (Tokyo). DNase I was obtained from Regeneration of spheroplasts. The spheroplast mixtures Sigma Chemical Co. (St. Louis). All other reagents were were diluted with R medium and plated on R medium of the highest grade available from commercial sources. containing 1.5% agar (R-solid medium). R medium con- taining 0.8% agar (R-soft medium) was overlaid onto the RESULTS surface of the R-solid medium.The numbers of colonies were determined after 3 to 5 days incubation at 30°C. The number of regenerated cells was calculated by deducting SpheroplastIn general,formationgram-negative bacteria are the numberof osmoicresistant cells fromthe numberof colonies on R medium. The frequency of regeneration was known to be converted into spherical bodies expressed as the ratio of the numberof regenerated cells to by a lysozyme-EDTA method.12) However, the numberof spheroplasts. spheroplasts of Z. mobilis (Z-6) could not be formed with this method. Since it is known Spheroplast fusion. Spheroplasts of two strains, Z-6-C fru~ and Z-6-C sue", were mixed and harvested by that the biosynthesis of bacterial cell walls is specifically inhibited by penicillins or gly- centrifugation at 3000rpm for lOmin at 4°C. They were washed with 10mM Tris-HCl, pH 7.2, containing 50mM cine,13) the organism was cultured in S medium CaCl2 and 20% sucrose. The washed spheroplasts were containing penicillin G or glycine to generate resuspended in a one-tenth volume of 10 mMTris-HCl, pH 7.2, containing 50mM CaCl2 and 33% polyethylene glycol spheroplasts; spherical cells were observed un- 6000 (PEG 6000). After incubation at 30°C for 60 min, the der a microscope. Spherical bodies began to suspension was diluted with Raffinose-R medium appear after 3 hr cultivation, and more than (R-medium containing 5%raffinose instead of glucose). 99.99% of the cells had been converted to a For the selection of fusants whose ability to ferment raffinose had been restored, Raffinose-R medium was 100r i n n n n n used. The suspension was spread on a 1.5% agar plate of Raffinose-R medium, and overlaid with the same medium containing 0.8% agar. After 14 days incubation at 30°C, colonies were isolated and fusants were examined as to their sugar fermentation properties. Analyses. Ethanol was quantitatively determined with a Hitachi Gas Chromatograph Model 163 equipped with a flame ionizing detector and a stainless steel column 0 100 200 00 0 1 0 0 D (3.2mmx2m) packed with Porapak Q (80 to 100mesh). The conditions for analysis were as follows: Carrier gas, Fig. 1. Effects of Concentrations of Penicillin G and nitrogen (40ml/min); temperature, 160~ 185°C for the Glycine on Spheroplast Formation of Z. mobilis Z-6. column, 230°C for the injection and detector; internal Spheroplasts of Z-6 were prepared by cultivation in S standard, isopropanol. mediumcontaining penicillin G and glycine at the con- Glucose, fructose, sucrose, melibiose and raffinose were centrations indicated at 30°C for lOhr. The numbers of analyzed and determined with a Liquid Chromatograph spheroplasts and osmotic resistant cells were calculated by equipped with a Shodex RI Model SE-ll and an Ionpak the method described in Materials and Methods. jii^ , KS-802 column. The conditions for analysis were as ratio of the numberof spheroplasts to the numberof total follows: Effluent, H2O(l.O ml/min); column temperature, 60°C. cells, percent; $p , ratio of the number of osmotic resistent cells to the number of total cells, percent. Spheroplast Fusion of Zymomonas 135 Fig. 2. Electron Micrograph of a Thin Section of Penicillin G-Induced Spheroplats of Z. mobilis Z-6 spherical form after 10hr. Figure 1 shows the effects of the concentrations of penicillin G and glycine, respectively, on the formation of spheroplasts of Z. mobilis. The optimum con- centrations were determined to be 100 units/ml for penicillin G and 3% for glycine, since the ratio of the number of osmotic resistant cells to the number of spheroplasts was lowest at that concentration. An electron micrograph of a thin section of a spheroplast of Z. mobilis is shown in Fig. 2. 1.0% Detached cell walls and exposed cytoplasmic 3.0% 5.0% (%) membraneswere observed on spheroplasts. 50100 Frequency of -, regeneration P mmmmtm^m fmmmmmmm |Wa:ffl=;¥ffl$S?;-ft-:-;-;-K-;-;-K-3Kxmxmx-mK^m l.ixlO"2 Regeneration and colony formation of Fig. 3. Effects of Concentrations of Penicillin G or spherop lasts Glycine on Regeneration of Z. mobilis Z-6. For the regeneration of spheroplasts of Spheroplasts of Z-6 were prepared by cultivation in S yeast and gram-positive bacteria, an osmotic mediumcontaining the indicated concentrations of penic- stabilizer such as 0.6n KC1,14) 0.5n NaCl,15) illin G or glycine.
Load more

Recommended publications
  • Distinct Single-Cell Morphological Dynamics Under Beta-Lactam Antibiotics
    Molecular Cell Article Distinct Single-Cell Morphological Dynamics under Beta-Lactam Antibiotics Zhizhong Yao,1,3 Daniel Kahne,1,4,* and Roy Kishony2,3,* 1Department of Chemistry and Chemical Biology 2School of Engineering and Applied Sciences Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA 3Department of Systems Biology 4Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA *Correspondence: [email protected] (D.K.), [email protected] (R.K.) http://dx.doi.org/10.1016/j.molcel.2012.09.016 SUMMARY in cell division, elongation, and shape maintenance (Spratt, 1975). It has been proposed that inhibition of crosslink forma- The bacterial cell wall is conserved in prokaryotes, tion by beta-lactams combined with misregulated cell-wall stabilizing cells against osmotic stress. Beta- degradation by PG hydrolases results in the accumulation of lactams inhibit cell-wall synthesis and induce lysis PG defects, which ultimately leads to cell lysis (Chung et al., through a bulge-mediated mechanism; however, 2009). little is known about the formation dynamics and The physical process of PG defect formation and subsequent stability of these bulges. To capture processes of lysis is poorly understood. Previous literature suggested a bulge-mediated process (Chung et al., 2009; Huang et al., different timescales, we developed an imaging 2008), and the reported rates of lysis have been loosely charac- platform combining automated image analysis with terized as slow and fast (de Pedro et al., 2002). However, in the live-cell microscopy at high time resolution. Beta- absence of systematic characterization of bulge-formation lactam killing of Escherichia coli cells proceeded dynamics and their variability across individual cells, it is unclear through four stages: elongation, bulge formation, whether lysis occurs uniformly within isogenic cell populations, bulge stagnation, and lysis.
    [Show full text]
  • The Effect of Spheroplast Formation on the Transformation Efficiency in Escherichia Coli Dh5α IRIS LIU, MARTHA LIU, KAREN SHERGILL Microbiology and Immunology, UBC
    Journal of Experimental Microbiology and Immunology (JEMI) Vol. 9:81-85 Copyright © April 2006, M&I, UBC The Effect of Spheroplast Formation on the Transformation Efficiency in Escherichia coli DH5α IRIS LIU, MARTHA LIU, KAREN SHERGILL Microbiology and Immunology, UBC Based on the observation that transformation in Escherichia coli is an inefficient process, it was hypothesized that transformation may be hindered by the presence of the outer membrane and the cell wall. To test this hypothesis, cells which lack both a cell wall and most of its outer membrane, termed spheroplasts, were created and transformed with pUC8 plasmid. Spheroplast formation was induced by incubating exponentially growing E. coli DH5α in ampicillin and sucrose. To transform these spheroplasts, the cells were subjected to a modified version of the Hanahan protocol, which involves treating the cells with calcium chloride. The results of this experiment suggest that spheroplasts can transform at 4.9 × 10-4. This is 100 fold more efficient than using the modified Hanahan protocol on wildtype E. coli DH5α cells. ________________________________________________________________ Competence is the ability of a bacterium to take up and physical aspects of the outer membrane hinders foreign DNA from the environment and the DNA uptake. If this is true, then removing the outer recombination or incorporation of this DNA is known membrane should increase transformation efficiency. as transformation (14). Although Escherichia coli is In the past, many groups have created strains of E. not considered to be a naturally competent organism, coli, such as ML-308, which have lost most their cell competence can be developed through various wall and only have remnants of an outer membrane (1, procedures.
    [Show full text]
  • Automated Image Analysis for Quantification of Filamentous Bacteria
    Syddansk Universitet Automated image analysis for quantification of filamentous bacteria Fredborg, M.; Rosenvinge, Flemming Schønning; Spillum, E.; Kroghsbo, S.; Wang, M.; Søndergaard, Teis Esben Published in: B M C Microbiology DOI: 10.1186/s12866-015-0583-5 Publication date: 2015 Document version Publisher's PDF, also known as Version of record Document license CC BY Citation for pulished version (APA): Fredborg, M., Rosenvinge, F. S., Spillum, E., Kroghsbo, S., Wang, M., & Sondergaard, T. E. (2015). Automated image analysis for quantification of filamentous bacteria. B M C Microbiology, 15, [255]. DOI: 10.1186/s12866- 015-0583-5 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 09. Sep. 2018 Fredborg et al. BMC Microbiology (2015) 15:255 DOI 10.1186/s12866-015-0583-5 METHODOLOGY ARTICLE Open Access Automated image analysis for quantification of filamentous bacteria Marlene Fredborg1,4*, Flemming S.
    [Show full text]
  • Final Report of the Lyme Disease Review Panel of the Infectious Diseases Society of America (IDSA)
    Final Report of the Lyme Disease Review Panel of the Infectious Diseases Society of America (IDSA) INTRODUCTION AND PURPOSE In November 2006, the Connecticut Attorney General (CAG), Richard Blumenthal, initiated an antitrust investigation to determine whether the Infectious Diseases Society of America (IDSA) violated antitrust laws in the promulgation of the IDSA’s 2006 Lyme disease guidelines, entitled “The Clinical Assessment, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and Babesiosis: Clinical Practice Guidelines by the Infectious Diseases Society of America” (the 2006 Lyme Guidelines). IDSA maintained that it had developed the 2006 Lyme disease guidelines based on a proper review of the medical/scientifi c studies and evidence by a panel of experts in the prevention, diagnosis, and treatment of Lyme disease. In April 2008, the CAG and the IDSA reached an agreement to end the investigation. Under the Agreement and its attached Action Plan, the 2006 Lyme Guidelines remain in effect, and the Society agreed to convene a Review Panel whose task would be to determine whether or not the 2006 Lyme Guidelines were based on sound medical/scientifi c evidence and whether or not these guidelines required change or revision. The Review Panel was not charged with updating or rewriting the 2006 Lyme Guidelines. Any recommendation for update or revision to the 2006 Lyme Guidelines would be conducted by a separate IDSA group. This document is the Final Report of the Review Panel. REVIEW PANEL MEMBERS Carol J. Baker, MD, Review Panel Chair Baylor College of Medicine Houston, TX William A. Charini, MD Lawrence General Hospital, Lawrence, MA Paul H.
    [Show full text]
  • Spheroplast-Mediated Carbapenem Tolerance in Gram-Negative
    bioRxiv preprint doi: https://doi.org/10.1101/578559; this version posted March 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Spheroplast-mediated carbapenem tolerance in Gram-negative 2 pathogens 3 4 Trevor Cross *1, Brett Ransegnola *1, Jung-Ho Shin 1, Anna Weaver 1, Kathy Fauntleroy 5 2, Mike VanNieuwenhze 3, Lars F. Westblade 2,4# and Tobias Dörr 1# 6 7 1 Weill Institute for Cell and Molecular Biology and Department of Microbiology, Cornell University, Ithaca, 8 NY, USA 9 2 Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA 10 3 Department of Molecular and Cellular Biochemistry and Department of Biology, Indiana University, 11 Bloomington, IN, USA 12 4 Division of Infectious Diseases, Department of Medicine, Weill Cornell Medicine, New York, NY, USA 13 14 *These authors contributed equally to this work 15 #To whom correspondence should be addressed: 16 [email protected] or [email protected] 17 18 Running title: Spheroplast-mediated carbapenem tolerance 19 20 Keywords: antibiotic, carbapenem, carbapenemase, Gram-negative, L- 21 form, meropenem, tolerance 22 23 bioRxiv preprint doi: https://doi.org/10.1101/578559; this version posted March 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 Abstract 25 Antibiotic tolerance, the ability to temporarily sustain viability in the presence of 26 bactericidal antibiotics, constitutes an understudied, yet likely widespread cause of 27 antibiotic treatment failure.
    [Show full text]
  • Horizontal Gene Transfers and Cell Fusions in Microbiology, Immunology and Oncology (Review)
    441-465.qxd 20/7/2009 08:23 Ì ™ÂÏ›‰·441 INTERNATIONAL JOURNAL OF ONCOLOGY 35: 441-465, 2009 441 Horizontal gene transfers and cell fusions in microbiology, immunology and oncology (Review) JOSEPH G. SINKOVICS St. Joseph's Hospital's Cancer Institute Affiliated with the H. L. Moffitt Comprehensive Cancer Center; Departments of Medical Microbiology/Immunology and Molecular Medicine, The University of South Florida College of Medicine, Tampa, FL 33607-6307, USA Received April 17, 2009; Accepted June 4, 2009 DOI: 10.3892/ijo_00000357 Abstract. Evolving young genomes of archaea, prokaryota or immunogenic genetic materials. Naturally formed hybrids and unicellular eukaryota were wide open for the acceptance of dendritic and tumor cells are often tolerogenic, whereas of alien genomic sequences, which they often preserved laboratory products of these unisons may be immunogenic in and vertically transferred to their descendants throughout the hosts of origin. As human breast cancer stem cells are three billion years of evolution. Established complex large induced by a treacherous class of CD8+ T cells to undergo genomes, although seeded with ancestral retroelements, have epithelial to mesenchymal (ETM) transition and to yield to come to regulate strictly their integrity. However, intruding malignant transformation by the omnipresent proto-ocogenes retroelements, especially the descendents of Ty3/Gypsy, (for example, the ras oncogenes), they become defenseless the chromoviruses, continue to find their ways into even the toward oncolytic viruses. Cell fusions and horizontal exchanges most established genomes. The simian and hominoid-Homo of genes are fundamental attributes and inherent characteristics genomes preserved and accommodated a large number of of the living matter.
    [Show full text]
  • Morphological and Ultrastructural Changes in Bacterial Cells As an Indicator of Antibacterial Mechanism of Action
    Cell. Mol. Life Sci. DOI 10.1007/s00018-016-2302-2 Cellular and Molecular Life Sciences REVIEW Morphological and ultrastructural changes in bacterial cells as an indicator of antibacterial mechanism of action 1 2 2 T. P. Tim Cushnie • Noe¨lle H. O’Driscoll • Andrew J. Lamb Received: 24 April 2016 / Revised: 21 June 2016 / Accepted: 28 June 2016 Ó Springer International Publishing 2016 Abstract Efforts to reduce the global burden of bacterial highlighting those morphological and ultrastructural chan- disease and contend with escalating bacterial resistance are ges which are consistently induced by agents sharing the spurring innovation in antibacterial drug and biocide same mechanism (e.g. spheroplast formation by peptido- development and related technologies such as photody- glycan synthesis inhibitors) and explaining how changes namic therapy and photochemical disinfection. Elucidation that are induced by multiple antibacterial classes (e.g. fil- of the mechanism of action of these new agents and pro- amentation by DNA synthesis inhibitors, FtsZ disruptors, cesses can greatly facilitate their development, but it is a and other types of agent) can still yield useful mechanistic complex endeavour. One strategy that has been popular for information. Lastly, recommendations are made regarding many years, and which is garnering increasing interest due future study design and execution. to recent technological advances in microscopy and a deeper understanding of the molecular events involved, is Keywords Antibiotic Á Antiseptic Á Disinfectant Á the examination of treated bacteria for changes to their Mode of action Á SEM Á TEM morphology and ultrastructure. In this review, we take a critical look at this approach.
    [Show full text]
  • Factors That Affect the Enlargement of Bacterial Protoplasts and Spheroplasts
    International Journal of Molecular Sciences Review Factors That Affect the Enlargement of Bacterial Protoplasts and Spheroplasts Hiromi Nishida Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan; [email protected]; Tel.: +81-766-56-7500 Received: 7 August 2020; Accepted: 25 September 2020; Published: 27 September 2020 Abstract: Cell enlargement is essential for the microinjection of various substances into bacterial cells. The cell wall (peptidoglycan) inhibits cell enlargement. Thus, bacterial protoplasts/spheroplasts are used for enlargement because they lack cell wall. Though bacterial species that are capable of gene manipulation are limited, procedure for bacterial cell enlargement does not involve any gene manipulation technique. In order to prevent cell wall resynthesis during enlargement of protoplasts/spheroplasts, incubation media are supplemented with inhibitors of peptidoglycan biosynthesis such as penicillin. Moreover, metal ion composition in the incubation medium affects the properties of the plasma membrane. Therefore, in order to generate enlarged cells that are suitable for microinjection, metal ion composition in the medium should be considered. Experiment of bacterial protoplast or spheroplast enlargement is useful for studies on bacterial plasma membrane biosynthesis. In this paper, we have summarized the factors that influence bacterial cell enlargement. Keywords: bacterial cell enlargement; microinjection; protoplasts; spheroplasts; metal ion composition; DNA replication; vacuole formation 1. Introduction Bacteria usually grow asexually, inheriting their DNA by clonal production (cell division). Therefore, these clones have the same genetic information. If clonal production were the only form of inheritance in bacteria, bacteria would display limited genetic variation. However, bacteria are so diverse that more than 99% are thought to be unknown at the species level.
    [Show full text]
  • Sonophotocatalysis Mediated Morphological Transition Modulates Virulence and Antibiotic Resistance in Salmonella Typhimurium
    Electronic Supplementary Material (ESI) for Environmental Science: Water Research & Technology. This journal is © The Royal Society of Chemistry 2020 Electronic Supplementary Information Sonophotocatalysis mediated morphological transition modulates virulence and antibiotic resistance in Salmonella Typhimurium A. P. Habeeb Rahman a, b, Ananyo Jyoti Misra a, b, Swagatika Panda c, Bhaskar Das d, Prachi Bhol a, Priti Sundar Mohanty a, b, Ashok J. Tamhankar b, e, Amrita Mishra b, Cecilia Stålsby Lundborg e, Suraj K. Tripathy a, b * a School of Chemical Technology, Kalinga Institute of Industrial Technology, Bhubaneswar, India b School of Biotechnology, Kalinga Institute of Industrial Technology, Bhubaneswar, India c ICMR-Regional Medical Research Center, Bhubaneswar, India dDepartment of Biotechnology and Medical Engineering, National Institute of Technology, Raurkela, India eDepartment of Public Health Sciences, Karolinska Institutet, Stockholm, Sweden * Corresponding author: S. K. Tripathy (E-mail: [email protected], Tel: +91-674-2725466) Synthesis of Fe-ZnO NPs: Supplementary Scheme. 1 Flowchart of Fe-ZnO NPs synthesis 8 7 6 5 L m / 4 U F C 0 1 3 g o l STm 2 STm SPI SB300 1 STm GFP SEH 0 0 20 40 60 80 100 120 Time (min) Fig. S1. Efficacy of SPC disinfection against different mutants of S. enterica. STm- antibiotic resistant mutant, STm SPI- Salmonella pathogenicity island induced STm, SB300- virulent strain of wild type S. Typhimurium, STm GFP- Green fluorescence protein tagged mutant, SEH- Hydrogen peroxide resistant mutant of S. Enteritidis. (Bacterial loading ≈ 107 CFU/mL, Fe-ZnO NPs loading = 200 mg/L, Temperature=35±2°C). Error bar indicates the standard deviation of replicates (n=3).
    [Show full text]
  • Optical Microscopy Reveals the Dynamic Nature of B. Pseudomallei Morphology During Β-Lactam Antimicrobial Susceptibility Testing Heather P
    McLaughlin et al. BMC Microbiology (2020) 20:209 https://doi.org/10.1186/s12866-020-01865-w RESEARCH ARTICLE Open Access Optical microscopy reveals the dynamic nature of B. pseudomallei morphology during β-lactam antimicrobial susceptibility testing Heather P. McLaughlin* , Julia Bugrysheva and David Sue Abstract Background: In Gram-negative species, β-lactam antibiotics target penicillin binding proteins (PBPs) resulting in morphological alterations of bacterial cells. Observations of antibiotic-induced cell morphology changes can rapidly and accurately differentiate drug susceptible from resistant bacterial strains; however, resistant cells do not always remain unchanged. Burkholderia pseudomallei is a Gram-negative, biothreat pathogen and the causative agent of melioidosis, an often fatal infectious disease for humans. Results: Here, we identified β-lactam targets in B. pseudomallei by in silico analysis. Ten genes encoding putative PBPs, including PBP-1, PBP-2, PBP-3 and PBP-6, were detected in the genomes of susceptible and resistant strains. Real-time, live-cell imaging of B. pseudomallei strains demonstrated dynamic morphological changes in broth containing clinically relevant β-lactam antibiotics. At sub-inhibitory concentrations of ceftazidime (CAZ), amoxicillin- clavulanic acid (AMC), and imipenem (IPM), filamentation, varying in length and proportion, was an initial response of the multidrug-resistant strain Bp1651 in exponential phase. However, a dominant morphotype reemerged during stationary phase that resembled cells unexposed to antibiotics. Similar morphology dynamics were observed for AMC-resistant strains, MSHR1655 and 724644, when exposed to sub-inhibitory concentrations of AMC. For all B. pseudomallei strains evaluated, increased exposure time and exposure to increased concentrations of AMC at and above minimal inhibitory concentrations (MICs) in broth resulted in cell morphology shifts from filaments to spheroplasts and/or cell lysis.
    [Show full text]
  • Liposidomycin, the First Reported Nucleoside Antibiotic Inhibitor of Peptidoglycan Biosynthesis Translocase I
    The Journal of Antibiotics (2019) 72:877–889 https://doi.org/10.1038/s41429-019-0241-5 SPECIAL FEATURE: REVIEW ARTICLE Liposidomycin, the first reported nucleoside antibiotic inhibitor of peptidoglycan biosynthesis translocase I: The discovery of liposidomycin and related compounds with a perspective on their application to new antibiotics Ken-ichi Kimura1 Received: 19 July 2019 / Revised: 17 September 2019 / Accepted: 17 September 2019 / Published online: 4 October 2019 © The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019 Abstract Liposidomycin is a uridyl liponucleoside antibiotic isolated from Streptomyces griseosporeus RK-1061. It was discovered by Isono in 1985, who had previously isolated and developed a related peptidyl nucleoside antibiotic, polyoxin, a specific inhibitor of chitin synthases, as a pesticide. He subsequently isolated liposidomycin, a specific inhibitor of bacterial peptidoglycan biosynthesis from actinomycetes, using a similar approach to the discovery of polyoxin. Liposidomycin has no cytotoxicity against BALB/3T3 cells but has antimicrobial activity against Mycobacterium spp. through inhibition of 1234567890();,: 1234567890();,: MraY (MurX) [phospho-N-acetylmuramoyl-pentapeptide transferase (translocase I, EC 2.7.8.13)]. Since the discovery of liposidomycin, several liposidomycin-type antibiotics, including caprazamycin, A-90289, and muraminomycin, have been reported, and their total synthesis and/or biosynthetic cluster genes have been studied. Most advanced, a semisynthetic compound derived from caprazamycin, CPZEN-45, is being developed as an antituberculosis agent. Translocase I is an interesting and tractable molecular target for new antituberculosis and antibiotic drug discovery against multidrug-resistant bacteria. This review is dedicated to Dr Isono on the occasion of his 88th birthday to recognize his role in the study of nucleoside antibiotics.
    [Show full text]
  • Author Section
    AUTHOR SECTION patients who may be treated in the outpatient setting with oral M antimicrobials from patients in whom hospitalization and parenteral therapy is appropriate. Over the past decade, dramatic escalation in antimicrobial resistance among common respiratory pathogens poses Macartney K.K. et al. Nosocomial respiratory syncytial virus infections: the obstacles to antibiotic choices.We review the microbiology of com- cost-effectiveness and cost-benefit of infection control. Pediatrics. 2000; munity-acquired pneumonia, and the therapeutic strategies that are 106(3) : 520-6.p Abstract: OBJECTIVE:To determine the cost- clinically and cost effective. effectiveness and cost-benefit of an infection control program to reduce nosocomial respiratory syncytial virus (RSV) transmission in Lyon W.R. et al. A role for trigger factor and an rgg-like regulator in the tran- a large pediatric hospital. DESIGN: RSV nosocomial infection (NI) scription, secretion and processing of the cysteine proteinase of Streptococcus was studied for 8 years, before and after intervention with a target- pyogenes. EMBO J. 1998; 17(21) : 6263-75.p Abstract: The abili- ed infection control program.The cost-effectiveness of the interven- ty of numerous microorganisms to cause disease relies upon the tion was calculated, and cost-benefit was estimated by a case-control highly regulated expression of secreted proteinases. In this study, comparison. SETTING: Children’s Hospital of Philadelphia, a 304- mutagenesis with a novel derivative of Tn4001 was used to identify bed pediatric hospital. PATIENTS: All inpatients with RSV infec- genes required for the expression of the secreted cysteine proteinase tion, both community- and hospital-acquired. INTERVENTION: (SCP) of the pathogenic Gram-positive bacterium Streptococcus Consisted of early recognition of patients with respiratory symp- pyogenes.
    [Show full text]