Laboratory Maintenance of Moraxella catarrhalis

Rachel Balder1, Eric R. Lafontaine1

1 University of Georgia, Department of Infectious Diseases, Athens, Georgia
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6B.1
DOI:  10.1002/9780471729259.mc06b01s11
Online Posting Date:  November, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Moraxella catarrhalis is a Gram‐negative bacterium that has recently emerged as the third leading cause of bacterial ear infections in children. This organism is also responsible for a variety of upper respiratory tract illnesses in adults, including ∼10% of all cases of respiratory exacerbations in patients with chronic obstructive pulmonary disease (COPD). There is interest in studying M. catarrhalis for vaccine development, and this unit provides guidelines for the laboratory maintenance of the organism. The three Basic Protocols presented in this unit describe how to culture and prepare M. catarrhalis cells for use in experiments pertaining to various biological aspects of this important respiratory pathogen. Curr. Protoc. Microbiol. 11:6B.1.1‐6B.1.11. © 2008 by John Wiley & Sons, Inc.

Keywords: Moraxella catarrhalis; growth; laboratory; biological assays; phase variation; viability

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of a Working M. catarrhalis Stock
  • Basic Protocol 2: Growth of M. catarrhalis on Solid Medium for Biological Assays
  • Basic Protocol 3: Growth of M. catarrhalis in Liquid Medium for Biological Assays
  • Support Protocol 1: Preparation of M. catarrhalis Frozen Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of a Working M. catarrhalis Stock

  Materials
  • Moraxella catarrhalis frozen stock vial (see protocol 4)
  • Todd Hewitt agar plates (see recipe)
  • Wire inoculating loop (see appendix 4A)
  • 37°C, 7.5% CO 2 incubator
  • Additional reagents and equipment for aseptic technique and streaking bacteria ( appendix 4A)

Basic Protocol 2: Growth of M. catarrhalis on Solid Medium for Biological Assays

  Materials
  • Moraxella catarrhalis working stock (see protocol 1)
  • Todd Hewitt agar plates (see recipe)
  • PBSG (see recipe)
  • Wire inoculating loop (see appendix 4A)
  • 37°C, 7.5% CO 2 incubator
  • 12 × 75–mm snap‐cap polypropylene tubes, sterile
  • Disposable inoculating loops, 10 µl (BD or equivalent)
  • Klett 802 calibrated test tubes (Scienceware/Bel‐Art Products) or spectrophotometer and appropriate cuvettes
  • Klett colorimeter with Klett color filter KS‐54
  • Additional reagents and equipment for aseptic technique and streaking bacteria ( appendix 4A)

Basic Protocol 3: Growth of M. catarrhalis in Liquid Medium for Biological Assays

  Materials
  • M. catarrhalis working stock (see protocol 1)
  • Todd Hewitt agar plates (see recipe)
  • PBSG (see recipe)
  • Todd Hewitt broth (see recipe)
  • Wire inoculating loop (see appendix 4A)
  • 37°C incubator (no CO 2 added) with shaker
  • 37°C, 7.5% CO 2 incubator
  • 12 × 75–mm snap‐cap polypropylene tubes, sterile
  • 300‐ to 500‐ml Nephelo culture flask, with 14 × 130–mm sidearm (Bellco Biotechnology or equivalent)
  • Klett colorimeter with Klett color filter KS‐54
  • Additional reagents and equipment for aseptic technique and streaking plates ( appendix 4A)

Support Protocol 1: Preparation of M. catarrhalis Frozen Stocks

  Materials
  • M. catarrhalis growing on agar plate (e.g., clinical isolate, purchased strain, or working stock needing to be restocked)
  • M. catarrhalis freezing medium (see recipe)
  • Disposable inoculating loops, 10 µl (BD or equivalent)
  • 2‐ml cryovials (Wheaton or equivalent)
  • −80°C freezer or liquid nitrogen tank
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Aebi, C., Stone, B., Beucher, M., Cope, L.D., Maciver, I., Thomas, S.E., McCracken, G.H. Jr., Sparling, P.F., and Hansen, E.J. 1996. Expression of the CopB outer membrane protein by Moraxella catarrhalis is regulated by iron and affects iron acquisition from transferrin and lactoferrin. Infect. Immun. 64:2024‐2030.
   Aebi, C., Lafontaine, E.R., Cope, L.D., Latimer, J.L., Lumbley, S.L., McCracken, G.H., Jr., and Hansen, E.J. 1998. Phenotypic effect of isogenic uspA1 and uspA2 mutations on Moraxella catarrhalis 035E. Infect. Immun. 66:3113‐3119.
   Attia, A.S. and Hansen, E.J. 2006. A conserved tetranucleotide repeat is necessary for wild‐type expression of the Moraxella catarrhalis UspA2 protein. J. Bacteriol. 188:7840‐7852.
   Attia, A.S., Lafontaine, E.R., Latimer, J.L., Aebi, C., Syrogiannopoulos, G.A., and Hansen, E.J. 2005. The UspA2 protein of Moraxella catarrhalis is directly involved in the expression of serum resistance. Infect. Immun. 73:2400‐2410.
   Balder, R., Hassel, J., Lipski, S., and Lafontaine, E.R. 2007. Moraxella catarrhalis strain O35E expresses two filamentous hemagglutinin‐like proteins that mediate adherence to human epithelial cells. Infect. Immun. 75:2765‐2775.
   Bonnah, R.A., Wong, H., Loosmore, S.M., and Schryvers, A.B. 1999. Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and lactoferrin receptor orf3 isogenic mutants. Infect. Immun. 67:1517‐1520.
   Bullard, B., Lipski, S.L., and Lafontaine, E.R. 2005. Hag directly mediates the adherence of Moraxella catarrhalis to human middle ear cells. Infect. Immun. 73:5127‐5136.
   Catlin, B.W. 1990. Branhamella catarrhalis: An organism gaining respect as a pathogen. Clin. Microbiol. Rev. 3:293‐320.
   Christensen, J.J. 1999. Moraxella (Branhamella) catarrhalis: Clinical, microbiological and immunological features in lower respiratory tract infections. APMIS 88:1‐36.
   Cripps, A.W., Otczyk, D.C., and Kyd, J.M. 2005. Bacterial otitis media: A vaccine preventable disease? Vaccine 23:2304‐2310.
   Doern, G.V. and Morse, S.A. 1980. Branhamella (Neisseria) catarrhalis: Criteria for laboratory identification. J. Clin. Microbiol. 11:193‐195.
   Furano, K. and Campagnari, A.A. 2003. Inactivation of the Moraxella catarrhalis 7169 ferric uptake regulator increases susceptibility to the bactericidal activity of normal human sera. Infect. Immun. 71:1843‐1848.
   Giebink, G.S., Kurono, Y., Bakaletz, L.O., Kyd, J.M., Barenkamp, S.J., Murphy, T.F., Green, B., Ogra, P.L., Gu, X.X., Patel, J.A., Heikkinen, T., Pelton, S.I., Hotomi, M., and Karma, P. 2005. Recent advances in otitis media. 6. Vaccine. Ann. Otol. Rhinol. Laryngol. 194:86‐103.
   Holm, M.M., Vanlerberg, S.L., Sledjeski, D.D., and Lafontaine, E.R. 2003. The Hag protein of Moraxella catarrhalis strain O35E is associated with adherence to human lung and middle ear cells. Infect. Immun. 71:4977‐4984.
   Holm, M.M., Vanlerberg, S.L., Foley, I.M., Sledjeski, D.D., and Lafontaine, E.R. 2004. The Moraxella catarrhalis porin‐like outer membrane protein CD is an adhesin for human lung cells. Infect. Immun. 72:1906‐1913.
   Jacobs, M.R., Bajaksouzian, S., Windau, A., Good, C.E., Lin, G., Pankuch, G.A., and Appelbaum, P.C. 2004. Susceptibility of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis to 17 oral antimicrobial agents based on pharmacodynamic parameters: 1998‐2001 U.S. Surveillance Study. Clin. Lab. Med. 24:503‐530.
   Karalus, R. and Campagnari, A. 2000. Moraxella catarrhalis: A review of an important human mucosal pathogen. Microbes Infect. 2:547‐559.
   Klein, J.O. 2000. The burden of otitis media. Vaccine 19:S2‐8.
   Klugman, K.P. 1996. The clinical relevance of in‐vitro resistance to penicillin, ampicillin, amoxycillin and alternative agents, for the treatment of community‐acquired pneumonia caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. J. Antimicrob. Chemother. 38:133‐140.
   Lafontaine, E.R., Wagner, N.J., and Hansen, E.J. 2001. Expression of the Moraxella catarrhalis UspA1 protein undergoes phase variation and is regulated at the transcriptional level. J. Bacteriol. 183:1540‐1551.
   Lipski, S.L., Akimana, C., Timpe, J.M., Wooten, R.M., and Lafontaine, E.R. 2007. The Moraxella catarrhalis autotransporter McaP is a conserved surface protein that mediates adherence to human epithelial cells through its N‐terminal passenger domain. Infect. Immun. 75:314‐324.
   Luke, N.R., Howlett, A.J., Shao, J., and Campagnari, A.A. 2004. Expression of type IV pili by Moraxella catarrhalis is essential for natural competence and is affected by iron limitation. Infect. Immun. 72:6262‐6270.
   Manninen, R., Huovinen, P., and Nissinen, A. 1997. Increasing antimicrobial resistance in Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in Finland. J. Antimicrob. Chemother. 40:387‐392.
   Mollenkvist, A., Nordstrom, T., Hallden, C., Christensen, J.J., Forsgren, A., and Riesbeck, K. 2003. The Moraxella catarrhalis immunoglobulin D‐binding protein MID has conserved sequences and is regulated by a mechanism corresponding to phase variation. J. Bacteriol. 185:2285‐2295.
   Murphy, T.F. 1996. Branhamella catarrhalis: Epidemiology, surface antigenic structure, and immune response. Microbiol. Rev. 60:267‐279.
   Murphy, T.F. and Loeb, M.R. 1989. Isolation of the outer membrane of Branhamella catarrhalis. Microb. Pathog. 6:159‐174.
   Murphy, T.F., Brauer, A.L., Grant, B.J., and Sethi, S. 2005. Moraxella catarrhalis in chronic obstructive pulmonary disease: Burden of disease and immune response. Am. J. Respir. Crit. Care Med. 172:195‐199.
   Murray, J.J., Emparanza, P., Lesinskas, E., Tawadrous, M., and Breen, J.D. 2005. Efficacy and safety of a novel, single‐dose azithromycin microsphere formulation versus 10 days of levofloxacin for the treatment of acute bacterial sinusitis in adults. Otolaryngol. Head Neck Surg. 133:194‐200.
   Pearson, M.M. and Hansen, E.J. 2007. Identification of gene products involved in biofilm production by Moraxella catarrhalis ETSU‐9 in vitro. Infect. Immun. 75:4316‐4325.
   Pearson, M.M., Lafontaine, E.R., Wagner, N.J., St. Geme, J.W. 3rd, and Hansen, E.J. 2002. A hag mutant of Moraxella catarrhalis strain O35E is deficient in hemagglutination, autoagglutination, and immunoglobulin D‐binding activities. Infect. Immun. 70:4523‐4533.
   Pearson, M.M., Laurence, C.A., Guinn, S.E., and Hansen, E.J. 2006. Biofilm formation by Moraxella catarrhalis in vitro: Roles of the UspA1 adhesin and the Hag hemagglutinin. Infect. Immun. 74:1588‐1596.
   Sethi, S. and Murphy, T.F. 2001. Bacterial infection in chronic obstructive pulmonary disease in 2000: A state‐of‐the‐art review. Clin. Microbiol. Rev. 14:336‐363.
   Sethi, S., Evans, N., Grant, B.J., and Murphy, T.F. 2002. New strains of bacteria and exacerbations of chronic obstructive pulmonary disease. N. Engl. J. Med. 347:465‐471.
   Singh, S., Cisera, K.M., Turnidge, J.D., and Russell, E.G. 1997. Selection of optimum laboratory tests for the identification of Moraxella catarrhalis. Pathology 29:206‐208.
   Timpe, J.M., Holm, M.M., Vanlerberg, S.L., Basrur, V., and Lafontaine, E.R. 2003. Identification of a Moraxella catarrhalis outer membrane protein exhibiting both adhesin and lipolytic activities. Infect. Immun. 71:4341‐4350.
   Verduin, C.M., Hol, C., Fleer, A., van Dijk, H., and van Belkum, A. 2002. Moraxella catarrhalis: From emerging to established pathogen. Clin. Microbiol. Rev. 15:125‐144.
   Wang, W., Pearson, M.M., Attia, A.S., Blick, R.J., and Hansen, E.J. 2007. A UspA2H‐negative variant of Moraxella catarrhalis strain O46E has a deletion in a homopolymeric nucleotide repeat common to uspA2H genes. Infect. Immun. 75:2035‐2045.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library