艰难梭菌孢子形成速率的体外测定

Adrianne N.  Edwards; Shonna M.  McBride

doi:10.21769/BioProtoc.2125

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Peer-reviewed

Determination of the in vitro Sporulation Frequency of Clostridium difficile

艰难梭菌孢子形成速率的体外测定

AE Adrianne N. Edwards email

SM Shonna M. McBride

发布: 2017年02月05日第7卷第3期 DOI: 10.21769/BioProtoc.2125 浏览次数: 8410

评审: Valentine V TrotterJoana AntunesAnonymous reviewer(s)

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Abstract

The anaerobic, gastrointestinal pathogen, Clostridium difficile, persists within the environment and spreads from host-to-host via its infectious form, the spore. To effectively study spore formation, the physical differentiation of vegetative cells from spores is required to determine the proportion of spores within a population of C. difficile. This protocol describes a method to accurately enumerate both viable vegetative cells and spores separately and subsequently calculate a sporulation frequency of a mixed C. difficile population from various in vitro growth conditions (Edwards et al., 2016b).

Keywords: Clostridium difficile (艰难梭菌)

Clostridium difficile infection (CDI) (艰难梭菌感染（CDI）)

Anaerobe (厌氧菌)

Spores (孢子)

Sporulation (孢子形成)

Ethanol resistance (耐乙醇性)

Background

Sporulation is a complex developmental process that results in the formation of a metabolically dormant spore. The physical properties of the C. difficile spore form provide intrinsic resistance to many environmental stresses and disinfectants, permitting its long-term survival outside of the host (reviewed in: Paredes-Sabja et al., 2014). To differentiate between the vegetative cells and spores of C. difficile, various techniques that take advantage of the physical and resistant properties of spores have been developed, including a short exposure to wet-heat or ethanol (Burns et al., 2010; Lawley et al., 2010; Edwards et al., 2014). However, these techniques may inadvertently cause long-term damage to the spores, depending on the strain of C. difficile tested, resulting in inaccurate recovery rates. Here, we describe an optimized method using a lower concentration of ethanol than previously described (40% less ethanol) to eliminate all vegetative cells within a heterogeneous C. difficile population without reducing the viability of spores. This technique provides highly reproducible and less variable results for quantifying C. difficile spore formation.

Materials and Reagents

Sterile inoculating loops (Grenier Bio One, catalog number: 731170 )
GeneMate 1.7 ml microcentrifuge tubes (BioExpress, catalog number: C-3260-1 )
Petri dishes (94 x 16 mm) (Grenier Bio One, catalog number: 633161 )
Glass test tubes with caps (18 x 150 mm) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 14-961-32 )
0.22 µm filter and syringe (for sterilization of taurocholate solutions) (CELLTREAT Scientific Products, catalog number: 229747 )
0.45 µm filter and syringe (for sterilization of D-fructose and L-cysteine solutions) (CELLTREAT Scientific Products, catalog number: 229749 )
Clostridium difficile strains of interest, including the isogenic parent strain (e.g., 630Δerm or R20291) as a reference strain or positive control, test strains, and a negative control strain that is unable to sporulate, preferably in the same isogenic background as the parent (e.g., a spo0A null mutant [Heap et al., 2007; Dawson et al., 2012; Deakin et al., 2012; Fimlaid et al., 2013; Mackin et al., 2013; Edwards et al., 2014; Edwards et al., 2016a])
95% ethanol (190 proof) (Decon Labs, catalog number: 2805SG )
Sterile water
Taurocholate (Sigma-Aldrich, catalog number: T4009 )
Brain heart infusion (BHI) (BD, catalog number: 237300 )
Yeast extract (BD, catalog number: 212730 )
Agar for solid medium (BD, catalog number: 214010 )
Bacto peptone (BD, catalog number: 211677 )
Proteose peptone (BD, catalog number: 211684 )
Tris base (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP152 )
Ammonium sulfate [(NH₄)SO₄] (Sigma-Aldrich, catalog number: A5132 )
L-cysteine (Sigma-Aldrich, catalog number: C7352 )
Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP358 )
Potassium chloride (KCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: P217 )
Sodium phosphate dibasic heptahydrate (Na₂HPO₄) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S373 )
Potassium phosphate monobasic (KH₂PO₄) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP362 )
D-fructose (Thermo Fisher Scientific, Fisher Scientific, catalog number: L96 )
10% (w/v) sodium taurocholate (see Recipes)
Pre-reduced BHIS agar (brain heart infusion supplemented with yeast extract; see Recipes)
Note: All media needs to be reduced before use. This is achieved by bringing plates or liquid medium into the anaerobic chamber at least 2 h for plates or overnight for liquid medium before use (for additional details, see Edwards et al., 2013).
Pre-reduced BHIS agar supplemented with 0.1% taurocholate
BHIS liquid medium (see Recipes)
70:30 sporulation agar (one plate per strain; see Recipes)
1x PBS (see Recipes)
20% D-fructose (see Recipes)
10% L-cysteine (see Recipes)

Equipment

Anaerobic chamber (Coy Type A or Type C Chamber)
Note: All steps are performed within the anaerobic chamber unless otherwise noted. Details on C. difficile cultivation as well as use and maintenance of an anaerobic chamber are described in (Edwards et al., 2013).
Spectrophotometer (Biochrom, model: CO8000 )
Autoclave

Procedure

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Edwards, A. N. and McBride, S. M. (2017). Determination of the in vitro Sporulation Frequency of Clostridium difficile. Bio-protocol 7(3): e2125. DOI: 10.21769/BioProtoc.2125.