Journal of Dairy Science
Volume 89, Issue 7 , Pages 2424-2427, July 2006

Technical Note: A Rapid Pulsed-Field Gel Electrophoresis Method for Analysis of Bifidobacteria

Department of Food Science, The Pennsylvania State University, University Park 16802

Received 4 October 2005; accepted 23 February 2006.

Article Outline

Abstract 

Pulsed-field gel electrophoresis (PFGE) is a widely used and highly discriminatory molecular typing method that has been applied to bifidobacteria. However, published PFGE protocols used with bifidobacteria require between 5 and 7 d to complete. A rapid PFGE method was developed that can be completed within 24h.

Key words: pulsed-field gel electrophoresis, Bifidobacterium

 

As the amount of scientific evidence supporting the reported health benefits of probiotics has risen, interest has grown in adding probiotic organisms to dairy products and to pharmaceutical preparations. A number of selection criteria have been proposed for probiotic microorganisms and, in addition to the various functional, technological, and safety criteria, appropriate methods must exist to differentiate one strain from another (Mattila-Sandholm et al., 2002; Champagne et al., 2005). Nucleic acid-based techniques have become increasingly important in differentiating and typing bacteria and in providing a means for evaluating inter-and intraspecies relatedness (Busch and Nitschko, 1999). Pulsed-field gel electrophoresis (PFGE) is a widely used and highly discriminatory molecular typing method based on comparison of fragment patterns of restriction-digested chromosomal DNA (Basim and Basim, 2001). Various researchers have applied PFGE to Bifidobacterium strains to estimate chromosome size (Bourget et al., 1993), to assess differences within and between human fecal samples (McCartney et al., 1996; Kimura et al., 1997; Rosberg-Cody et al., 2004), and to differentiate strains (Roy et al., 1996; Simpson et al., 2003; Yeung et al., 2004). However, reported PFGE protocols used with bifidobacteria are not conducive to routine analysis, requiring between 5 and 7 d to complete the assay. The objective of this research was to develop a PFGE protocol that could be applied to bifidobacteria and that could be completed within 24h.

Twelve strains of Bifidobacterium were obtained from the ATCC (American Type Culture Collection, Manassas, VA) and the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, the German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany), and 22 samples of Bifidobacterium were obtained from 6 commercial starter culture companies (Table 1). All strains were identified as Bifidobacterium by PCR amplification of a region of 16S rDNA based on the method of Kaufmann et al. (1997). Commercial strains were identified at the species level using PCR primers under the conditions described by Matsuki et al. (1999), Ventura et al. (2001), and Ventura and Zink (2002). Suspensions of strains from ATCC, DSMZ, and from commercial starter culture companies (4 strains: 2 Bifidobacterium animalis ssp. lactis, 1 Bifidobacterium infantis, and 1 Bifidobacterium longum) were prepared and separated into duplicate aliquots to compare the typical and modified protocols. All strains were then evaluated using the modified protocol. Comparisons of the typical and modified methods were replicated twice. The modified protocol was replicated with all strains at least in triplicate.

Table 1. Strains of Bifidobacterium spp. evaluated in this study
Strain name1Strain identity
ATCC 15696Bifidobacterium bifidum
ATCC 15697Bifidobacterium infantis
ATCC 15698Bifidobacterium breve
ATCC 15700B. breve
ATCC 15707Bifidobacterium longum
ATCC 15708B. longum
ATCC 25527Bifidobacterium animalis ssp. animalis
ATCC 27536B. animalis ssp. lactis
ATCC 27672B. animalis ssp. animalis
ATCC 27686Bifidobacterium choerinum
ATCC 29521B. bifidum
DSMZ 10140B. animalis ssp. lactis
B. animalis ssp. lactis (20 commercial strains)
B. infantis (1 commercial strain)
B. longum (1 commercial strain)

1ATCC = American Type Culture Collection (Manassas, VA); DSMZ = Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany).

For PFGE, strains were streaked onto liver lactose agar (35g/L liver infusion broth, 10g/L lactose, 10g/L trypticase peptone, 2g/L sodium chloride, 15g/L agar; Enumeration Procedures for Probiotics, Lyoferm, Inc., Indianapolis, IN) and incubated anaerobically for 3 d at 37° C in an anaerobic incubator (5mmHg vacuum pressure; anaerobic mixed gas: 10% carbon dioxide, 5% hydrogen, 85% nitrogen; VWR, West Chester, PA). A single colony was transferred to 10mL of liver lactose broth and incubated anaerobically overnight until turbid. A 2.0-mL aliquot was centrifuged for 10min at 14,000×g to pellet the cells, which were then washed once with 2.0mL of 100mM Tris, 100mM EDTA buffer (pH 7.6) and resuspended in 600μL of this buffer. This suspension was separated into duplicate 160-μL aliquots to compare the typical and modified PFGE protocols. To the 160μL of cell suspension used with the typical protocol was added an additional 50μL of the resuspension buffer to account for the dilution effect of adding lysis reagents during plug preparation with the modified PFGE protocol.

The “typical” protocol selected was based on that described by Simpson et al. (2002, 2003), which is representative of the PFGE methods found in the bifidobacteria literature. Briefly, the cell suspension was mixed with an equal volume of 2% InCert agarose (Cambrex, Rockland, ME) prepared in 0.125 M EDTA (pH 7.6) and dispensed into disposable plug molds (10×5×1.5mm; Bio-Rad, Hercules, CA). The plugs were incubated in 1mL of 1 M NaCl, 6mM Tris-HCl, 100mM EDTA, 1% Sarkosyl buffer (pH 7.6; Sigma, St. Louis, MO) with 10mg/mL lysozyme (Sigma) and 500 units/mL mutanolysin (Promega Corp., Madison, WI) at 37°C for 18h. The plugs were then incubated in fresh Sarkosyl buffer with 0.8mg/mL proteinase K (Sigma) at 37°C for 18h. The proteinase K buffer was refreshed and the plugs were then incubated for an additional 18h. The plugs were washed twice with 1mM phenylmethylsulfonyl fluoride (PMSF; Sigma) in 10mM Tris-HCl, 1mM EDTA (pH 8.0) at 37°C for 60min in a shaking water bath (New Brunswick Scientific, Edison, NJ). Two slices (1-mm wide) were prepared from the plugs and washed 3 times in 1mL of 10mM Tris-HCl, 0.1mM EDTA (pH 8.0) for 15min at room temperature. The slices were preincubated at 4°C for 30min in 100μL of the appropriate restriction endonuclease buffer. They were then transferred to 100μL of a fresh restriction digest mixture containing 30 units of XbaI or SpeI and incubated at 37°C for 18h.

To the cell suspension were added 40μL of lysozyme solution (100mg/mL) and 10μL of proteinase K solution (20mg/mL). This mixture was immediately combined with an equal volume of 1.6% InCert agarose prepared in 0.1% SDS (International Biotechnologies, Inc., New Haven, CT) and dispensed into disposable plug molds. Lysis was performed by incubating the plugs in 1.5mL of 0.5 M EDTA, 1% Sarkosyl buffer (pH 9.0) with 4mg/mL lysozyme and 200 units/mL mutanolysin at 55°C for 90min; the plugs were then incubated in fresh Sarkosyl buffer with 0.5mg/mL proteinase K at 55°C for 60min. The plugs were washed in preheated sterile distilled water at 50°C for 15min, then in 3 changes of preheated 10mM Tris, 1mM EDTA buffer (pH 7.6) at 50°C for 15min in a shaking water bath at 75rpm. Two slices were prepared from the plugs and incubated in 100μL of a restriction digest mixture with 30 units of XbaI or SpeI for 2h at 37°C.

Electrophoresis was performed on 1.0% SeaKem Gold agarose gel (Cambrex; Michaud et al., 2001) using 0.5×TBE buffer (45mM Tris, 45mM boric acid, 1mM EDTA, pH 8.0). Slices from the traditional and modified methods, from the same initial cell preparation, were loaded in adjacent lanes for comparison. A lambda ladder (Bio-Rad) was included as a molecular weight marker. Electrophoresis was performed using a CHEF Mapper System (Bio-Rad). Switch times were increased linearly from 0.19 to 35.38s for 13.9h, with an angle of 120° at 6 V/cm and 14°C. Gels were stained with a solution of ethidium bromide (0.4mg/L; Promega) for 1h, then destained for 2h. Restriction patterns were visualized on a UV transilluminator (302nm), and images were captured using an AlphaImager 3300 Gel Documentation System (Alpha Innotech Corp., San Leandro, CA) and saved as .TIFF files for future analysis.

Banding patterns obtained for each strain from both the typical and modified methods were identical (Figure 1), confirming the suitability of this rapid method with samples of bifidobacteria.

  • View full-size image.
  • Figure 1. 

    Pulsed-field gel electrophoresis comparing the traditional and rapid protocols, digested with XbaI. M: Lambda molecular weight marker; lanes with “a” were performed with the traditional method, and lanes with “b” were performed with the rapid method; lanes 1a/b: Bifidobacterium breve ATCC 15698; lanes 2a/b: B. breve ATCC 15700; Lanes 3a/b: Bifidobacterium longum ATCC 15707; Lanes 4a/b: B. longum ATCC 15708; Lanes 5a/b: Bifidobacterium animalis ssp. lactis (commercial strain).

Several modifications to the typical protocol were responsible for reducing the time needed to perform this technique. Most of the reduction in time for this method was associated with shorter incubation times during lysis, washing, and restriction.

Bifidobacteria are typically more difficult to lyse than gram negative bacteria, and PFGE protocols with bifidobacteria generally use long lysis times (18 to 72h). However, to reduce the lysis time, lysis reagents may be added to the initial cell suspension, then immediately combined with agarose to prevent undesirable shearing or degradation of the DNA, as recommended by Chang and Chui (1998). By adding lysozyme and proteinase K directly to the bifidobacteria cell suspension, incubation time in the lysis buffer with lysozyme and mutanolysin could be reduced to 90min. A higher incubation temperature (55°C) was also used, which is closer to the temperature for maximum activity of the mutanolysin (Yokogawa et al., 1974). When the incubation time in this step was varied from 90min to 18h, no difference in the intensity of the bands was observed. Although many of the bifidobacteria strains lysed without the addition of mutanolysin, it was necessary to add mutanolysin (200 units/mL) to the buffer to ensure complete lysis of all 34 strains examined. In an effort to reduce reagent costs associated with the modified method, subsequent experiments using a lower concentration of mutanolysin (40 units/mL) were performed, which resulted in complete lysis of 33 of the 34 strains of bifidobacteria within 1.5h. Therefore, this lower mutanolysin concentration of 40 units/mL has been used in our laboratory for routine PFGE analyses.

The incubation of the plugs in proteinase K buffer was also reduced from between 12 and 50h, as in traditional bifidobacterial protocols, to 60min in the rapid protocol. When this step was varied between 1 and 4h, there was no difference in band intensity among the strains examined.

The washing steps were also modified from those of traditional PFGE protocols. Most protocols with bifidobacteria use PMSF, a serine protease inhibitor, to inactivate proteinase K, followed by a series of washes to remove the PMSF (McCartney et al., 1996; Roy et al., 1996; Sanders et al., 1996; Kimura et al., 1997; Crittenden et al., 2001; Ventura and Zink, 2002; Yeung et al., 2002; Gueimonde et al., 2004; Mättö et al., 2004). Some protocols have extensively washed plugs to remove the proteinase K rather than inactivating it (Grand et al., 2003; O’Riordan and Fitzgerald, 1997). However, washing steps often will take up to 30h in traditional methods. The rapid PFGE methods for Escherichia coli and Campylobacter, presented by Gautom (1997) and Michaud et al. (2001), respectively, suggested preheating the water or buffer and using a shaking water bath to shorten the time for this step. This procedure was applied in the rapid method for bifidobacteria—4 washes of 15min each were performed with water and Tris, EDTA buffer at 50°C—without negatively affecting the subsequent restriction of the DNA.

Restriction was also shortened from a traditional overnight incubation to 2h without increasing the amount of enzyme used. Other rapid methods have used digest times between 90min and 3h (Matushek et al., 1996; Gautom, 1997; Chang and Chui, 1998; Michaud et al., 2001). A long incubation time was not necessary with samples prepared according to the rapid method based on the identical patterns obtained when digestion occurred over 18h.

A rapid PFGE method was developed for bifidobacteria that may be completed, from a turbid tube of culture media to a picture of a gel, within 24h. Although no effort was made to determine the minimum incubation times necessary at each step, the times reported here have been used successfully with our collection of bifidobacteria strains, suggesting that the long times used in traditional methods are unnecessary. This method should allow PFGE to be more readily applied to bifidobacteria isolates in the dairy and supplement industries.

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PII: S0022-0302(06)72315-3

doi:10.3168/jds.S0022-0302(06)72315-3

Journal of Dairy Science
Volume 89, Issue 7 , Pages 2424-2427, July 2006

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