differentiation of bifidobacteria by use of pulsed-field gel electrophoresis and polymerase chain...

ELSEVIER International Journal of

Food Microbiology 29 (1996) 11-29

International Journal of Food Microbiology

Differentiation of bifidobacteria by use of pulsed-field gel electrophoresis and polymerase chain reaction

Denis Roy *, Pierre Ward, Guy Champagne

Food Research and Development Centre, Agriculture Canada 3600, Casavant Boulevard West, Saint-Hyacinthe, Quebec, Canada J2S 8E3

Received 27 June 1994; accepted 6 January 1995

Abstract

Several different genomic fingerprints can be obtained from various commercially-important species of Bifi&bactetium using pulsed-field gel electrophoresis (PFGE) following digestion of DNA with Xbal and S&I. Four different genomic fingerprintings were discernible for reference strains of Bifidobacterium animalis, five for B. bifidum, three for B. breve, five for B. infantis and three for B. longum. Standard commercially-available industrial strains of B. animalis are identical to the reference strain ATCC 27536, previously isolated from chicken feces. There was more genomic heterogeneity among industrial strains of B. longum, in that only one gave profiles similar to the type strain of this species (ATCC 15707). The other 14 commercially-available strains of B. longum (mainly isolated from Japanese commercial preparations) were divided into four new molecular types based on their PFGE patterns. The PFGE method indicated that only five distinct strains of B. fongum and one strain of B. animafis are used in commercial preparations. Additionally, the use of polymerase chain reaction amplification of portions of 16s rDNA provides a highly specific technique to discriminate between the species B. breve, B. infantis and B. longum.

Keywords: Bifidobacteria; Pulsed-field gel electrophoresis; Polymerase chain reaction

1. Introduction

The genus Bifidobacterium includes 29 species, and 10 of which are of human

origin. The most suitable species of human origin used for the production of

* Corresponding author. Fax: (514)773-8461

0168-1605/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDIO168-1605(95)00013-5

12 D. Roy et al. /ht. J. Food Microbiology 29 (1996) I1 -29

fermented dairy products are Bifidobacterium longum, B. breve, B. bifidum and B. infantis. The species B. animal& previously isolated from warm-blooded animals, has also been found in fermented dairy products (Bonaparte and Reuter, 1991; Biavati et al., 1992). The increasing number of commercial strains of bifidobacteria used in the food industry requires reliable methods for characterization and control, since it must be ensured that they are of human origin (Biavati et al., 1992).

Several methods, based on phenotypic characterization, have been proposed for the identification of dairy-related bifidobacteria (Chevalier et al., 1990; Roy and Ward, 1990; Yaeshima et al., 1992). Commercial strains of B. animalis may be differentiated from those of B. longum by using phenotypic characteristics and @galactosidase electrophoretic patterns (Roy et al., 1994). However, numerical analysis of carbohydrate fermentation patterns and enzymatic activity profiles indicates that, among species of human origin, B. longum and B. infantis can not be differentiated on the basis of a large number of phenotypic characteristics (Bahaka et al., 1993). DNA-DNA hybridization studies have also demonstrated that the levels of DNA relatedness between these two species are very similar (Lauer and Kandler, 1983; Bahaka et al., 1993).

Few studies have been published on the use of other molecular methods such as DNA fingerprinting and ribotyping to compare strains or species of bifidobacteria. Recently, the genomes of five B. breve strains were compared by restriction endonuclease analysis (Bourget et al., 1993). Pulsed-field gel electrophoresis (PFGE) combined with controlled restriction by rare-cutting endonucleases has been used for strain differentiation and chromosome size estimation in lactic acid bacteria (Bourget et al., 1993; Tanskanen et al., 1990). The genomes of bifidobacteria have a G + C content varying from 55 to 64% (Scardovi, 1986). Restriction enzymes with recognition sequences rich in A and T nucleotides such as XbaI (TCTAGA) and spe1 (ACTAGT) may be used to produce few large DNA fragments that can be resolved by PFGE (McClelland et al., 1987).

Yamamoto et al. (1992) have synthesized DNA probes based on portions of 16s rRNA specific for five Bifidobacterium species which are often detected in human feces. Frothingham et al. (1993) also noted that rDNA sequences may be useful for the characterization of bifidobacteria. The sequence analysis of 16s rDNA may also help to define species differences between B. longum and B. infantis. This molecule can be used to develop highly specific oligonucleotide primers which facilitate the unequivocal identification of microorganisms using the polymerase chain reaction (PCR).

The aim of this study was to determine the PFGE patterns of XbaI and SpeI digests of genomic DNA of dairy-related bifidobacteria (B. animalis, B. bifidum, B. brece, B. infantis and B. longurn) obtained from commercial preparations and culture collections to determine the origin of commercial strains of B. animalis and B. longum. Analysis of published 16s rDNA sequences allowed the design of specific DNA primers for use in PCR-amplification to differentiate strains of B. brelle, B. infantis and B. longum.

D. Roy et al. /ht. J. Food Microbiology 29 (I 996) I I-29 13

2. Materials and methods

2.1. Bacterial strains and cultivation

Strains of bifidobacteria obtained from commercial preparations (dairy products and freeze-dried cultures) are listed in Table 1. Twenty human strains of bifidobacteria isolated from child feces and adult intestine by Bahaka et al. (1993) are also listed in Table 1. Other bifidobacteria and lactic acid bacteria were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and the Deutsche Sammlung von Mikroorganismen (DSM, Gottingen, Germany) These strains were freeze dried in skim milk (20% w/w) and sucrose (5% w/w). Lactobacilli MRS broth (Difco Laboratories, Detroit, MI, USA)) supplemented with 0.05% r_-cysteine-HCl was used to rehydrate the freeze-dried microorganisms, and recovered strains were subcultured twice. Active cultures were incubated for 18 h at 37°C in an anaerobic chamber (Anaerobic system, Forma Scientific, Marietta, OH, USA) with 5% CO,, 10% H, and 85% N, gas atmosphere.

2,2. Biochemical tests

Enzymatic activity profiles and carbohydrate fermentation experiments were determined according to Roy and Ward (1990). The presence of fructose-6-phos- phate phosphoketolase was detected according to Chevalier et al. (1991).

2.3. DNA extraction

DNA from bacterial strains was isolated according to the procedures of Klaen- hammer (1984) for the simultaneous utilization of mutanolysine and lysozyme, and the procedures of Ausubel et al. (1991) for the extraction with CTAB/NaCl. The concentration of purified DNA was determined by absorbance at 260 nm.

2.4. Preparation of genomic DNA

Cells corresponding to 15-ml culture samples in MRS medium supplemented with 0.05% L-cysteine-HCI in stationary phase of growth (18-24 h, 37°C) were harvested by centrifugation for 20 min at 1700 X g, washed three times with 15 ml of 1M NaCl-10 mM Tris-HCI (pH 7.6), and resuspended in 750 ~1 of the same solution. Different dilutions of this suspension were prepared in the same solution. An aliquot (150 ~1) of the appropriate dilutions was vigorously mixed with an equal volume of 1.5% pulsed-field electrophoresis (PFE) licensed low-melting-point agarose (Beckman Instruments, Inc., Palo Alto, CA, USA) before solidifying in molds for 20 min at 4°C.

The embedded cells in agarose blocks were digested in situ with EC buffer (6 mM Tris chloride, pH 7.6, 1M NaCI, 100 mM EDTA, pH 7.6, 1% Sarkosyl, 1 mg/ml of lysozyrne) for 18 h at 37°C. The digested blocks were incubated in 0.5 M EDTA (pH 9.0) with 1% Sarkosyl and 1 mg/ml of proteinase K for 24 h at 50°C.

14

Table 1

D. Roy ei al. /ht. J. Food Microbiology 29 (1996) 1 I-29

Designation and origin of bifidobacterial strains used in this study

Designation Identified as a Isolated by

FRDC ’ FRDC

FRDC

FRDC

FRDC

G. Reuter H6

G. Reuter B15

G. Reuter B17

G. Reuter B18

G. Reuter BlY

G. Reuter B22

FRDC

FRDC

FRDC

FRDC FRDC

FRDC

CFAR 170

CFAR 171

CFAR 115

CFAR 116 CFAR 117

CFAR 169

CFAR 123

CFAR 63 CFAR 122

CFAR 118

CUETM 89-l 71 CUETM 89-172

CUETM 89-174

CUETM 89-186

CUETM 89-193

CUETM 89-216

CUETM 89-239

CUETM 89-245

CUETM 89-247

CUETM 89-257

CUETM 89-259

CUETM 89-260 CUETM 89-263 CUETM 89-267 CUETM 89-268 CUETM 89-276 CUETM 89-281 CUETM 89-287

CUETM 89-290

Isolated from

RW-003 RW-004

RW-005

RW-006

RW-011 RW-013

RW-014

RW-015

RW-016

RW-017

RW-018

RW-012 RW-010

RW-001

RW-002

RW-008

RW-009

RW-019

RW-020

RW-021

RW-022 RW-023

RW-024 RW-025

RW-026

RW-027

RW-028

89-171

89-172

89-174

89-186 89-193

89-216

89-239

89-245 89-247

89-257

89-259 89-260 89-263 89-267 89-268 89.276 89-281 89.287

89-290

B. animalis B. animalis B. animalis B. animalis B. animalis B. animalis B. animalis B. animalis B. animalis B. animalis B. animalis A. bifidum B. brew B. longum B. longum B. longum B. longum B. longum B. longum B. longum B. longum B. longum B. longum B. longum B. longum B. longum B. longum B. lorzgum B. longum B. adolescentis B. longum B. longum B. longum B. longum B. longum B. longum B. longum B. longum B. iongum B. longum B. longurn B. longum B. longum B. longum B. longurn B. longum

Commercial preparation

Fermented milk

Fermented milk


Commercial preparation Fermented milk

Fermented milk

Fermented milk

Fermented milk

Fermented milk

Fermented milk



Commercial preparation Commercial preparation






Commercial preparation Commercial preparation






Child feces ?

Child feces

Child feces

Child feces

Adult intestine

Child feces

Child feces

Child feces

Child feces

Child feces

Child feces Child feces Child feces Child feces Child feces Child feces Child feces

Child feces -

” Identified by numerical analysis according to Roy et al. (1994).

’ FRDC, Food Research and Development Centre, Agriculture Canada, St. Hyacinthe, Quebec,

Canada; G. Reuter, Freie Universitat Berlin, Germany; CFAR, Centre for Food and Animal Research, Ottawa, Ontario, Canada: CUETM, Collection Unit6 Ecotoxicologie, Villeneuve d’Ascq, France.

D. Roy et al. /ht. J. Food Microbiology 29 (1996) II -29 15

This latter step was repeated and the blocks were then treated twice for 1 h with 1 mM phenylmethylsulfonyl fluoride in TE (10 mM Tris chloride (pH 8.01, 1 mM EDTA), washed three times for 1 h at ambient temperature in TE buffer. The digested blocks could then be stored for several weeks at 4°C in 0.5 M EDTA (pH 8.0) - 1% Sarkosyl.

2.5. Digestion of DNA

Agarose blocks containing DNA were cut into slices (3 x 3 mm). Each slice was washed three times for 1 h at ambient temperature in TE buffer, placed in a sterile microcentrifuge tube containing 250 ~1 of restriction enzyme buffer and incubated for 18 h at 37°C with 30 U of XZxzI or $x1 (Boehringer Mannheim Canada, Inc., Laval, PQ, Canada).

2.6. Pulsed-field gel electrophoresis (PFGE)

Agarose gel 1% (LE Agarose, Beckman Instruments) was loaded with digested DNA. Samples were electrophoresed by using transverse alternating field electrophoresis (TAFE; Geneline II, Beckman Instruments) in 0.25 X TAE buffer (1 x TAE: 40 mM Tris-acetate, 1 mM EDTA, pH 8.0) at 15°C. The running condition was: (stage 1) 2 s pulse for 6 h at 350 mA; (stage 2) 5 s for 5 h at 370 mA; (stage 3) 10 s for 4 h at 390 mA; (stage 4) 15 s for 4 h at 410 mA; (stage 5) 30 s for 3 h at 430 mA; and (stage 6) 90 s for 2 h at 450 mA. Gels were stained for 45 min with 0.5 mg/l ethidium bromide, washed for 1 h with demineralized water, viewed by UV transillumination, and photographed using Polaroid 57 film. Molecular weight standards were included with each electrophoresis. The estimation of the size of the fragments and the comparison of patterns belonging to different strains were done using lambda DNA ladder and Saccharomyces cerevisiue chromosome (Boehringer Mannheim) as molecular size standards. DNA of different strains were analyzed 2 to 3 times.

Table 2 Synthesized primer pairs and their nomenclature

Primer Sequence Tm (“0 PCR Target strain product

(bp)

BreU3 5’.CTCCAGCTCGACTGTCGC 56.8 811 B. brece

BeL4 S-GCACT’ITGTG7TGAGTGTACCI-ITCG 58.0

InfU5 S-CCATCKTGGGATCGTCGG 55.0 565 B. infantis

InfL6 5’-TATCGGGGAGCAAGCGTGA 56.4 B.indicum

LonU7 S-GCCGTATCTCTACGACCGTCG 56.1 567 B. longum

LonL8 S-TATCGGGGAGCAAGCGAGAG 56.7 B. pseudolongum

I6 D. Roy et al. /ht. .I. Food Microbiology 29 (1996) I1 -29

439 489 B.breve ATCC 15700 TTTGTTAGGGAGCAAGGCACTTTGTGTTGAGTGTACCTTTCGAATAAGCA B.longum ATCC 15707 TTTATCGGGGAGCAAGmCG---AGAGT-GAGTTTACCCGTTGAATAAGCA B.infantis ATCC 15697 TTTATCGGGGAGCAAG-CG---TGAGT-GAGTTTACCCXTTGAATXAGCA

*t* l l ******** l * t. l *** **** * l *** t***

971 1021 B.breve ATCC 15700 GGCTTGACATGTTCCCGACGATCCCAGAGATGGGGTTTCCCTTCGGGGXG B.longum ATCC 15707 GGCTTGACATGTTCCCGACGGTCGTAGAGATACGGCXTCCCTTCGGGGCG B.infantis ATCC 15697 GGCTTGACXTGTTCCCGACGATCCCAGAGATG~GXXXXTCCTTCGGGXCG

***tt*t* l ********** ** **t*** l t******* * 1226 1276

B.breve ATCC 15700 AATGGCCGGTACAACGGGATGCGACAGTGCGAGCTGGAGCGGATCCCTGA B. longum ATCC 15707 AATGGCCGGTACAACGGGATGCGACGCGGCGACGCGGAGCGGATCCCTGA B.infantis ATCC 15697 AATGGCCGGTACAACGGGATGCGACGCGGCGACGCGGAGCGGATCCCTGA

l ******t*******t*+*t***** l *** ******+t**+**t*

BreL4 LonL8 InfL6

LonU7 InfUS

BrelJ3

Fig. 1. Partial 16s rDNA sequences of three Bifidobacterium spp. The target regions for species-specific

oligonucleotide probes are underlined. Numbers are based on the sequences (Genbank data library

accession numbers MS8731, M58739, M58738). Asterisk shown conserved nucleotides among the three

species.

2.7. Primers

Oligonucleotide primer pairs are shown in Table 2, and the corresponding regions in the 16s rDNA of B. breve, B. infuntis and B. longum are shown in Fig. 1. Primers BreU3, InfU5, and LonU7 are 5’-3’ nucleotides sequences complementary to the partial 16s rDNA sequences shown in Fig. 1. Primer BreU3, was previously described by Frothingham et al. (1993). Primers InfU5 and I_onU7 were chosen by comparison of 25 partial 16s rDNA sequences of Bifidobacterium spp. obtained from Genbank and from the Ribosomal Database Project. Primers BreL4, LonL8 (length-modified) and InfL6 are sequences complementary to the respective probes PBR, PIN and PLO described by Yamamoto et al. (1992). Multiple sequences were aligned using the CLUSTAL program of PCGENE (Intelligenetics, Mountain View, CA, USA). PCR primers were chosen using the PCRPLAN program of PCGENE. All synthetic oligonucleotide primers were purchased from General Synthesis and Diagnostics, Toronto, Ontario Canada.

2.8. Polymerase chain reaction (PCR)

Optimized concentrations for PCR (50 /*l total volume) were 0.25 FM primers; 200 PM each dATP, dCTP, dGTP and dTTP (Pharmacia LKB Biotechnology); 1 X PCR reaction buffer from Boehringer Mannheim (10 mM Tris-HCl; 1.5 mM MgCl,; 50 mM KCl; pH 8.3) and 1 unit of Taq DNA polymerase (Boehringer Mannheim). Between 250 and 500 ng of DNA were used for PCR reactions. Samples were covered with 75 ~1 of mineral oil and subjected to PCR on a DNA thermal Cycler (Perkin-Elmer Cetus Corp., Norwalk, CT, USA). Template DNA and oligonucleotide primers were initially heated at 95°C for 3 min and then cooled at 56°C. PCR reaction mixture (PCR buffer, dNTPs and Taq polymerase) were added at this temperature. PCR amplification was run for 40 cycles under the following conditions: denaturation at 95°C for 30 s, primer annealing at 56°C for 30 s and DNA extension at 73°C for 1 min. After cycling, 10 ~1 of each PCR reaction

D. Roy et al. /ht. J. Food Microbiology 29 (1996) II-29 17

mixture was run on a 2% agarose gel in 0.5 X TBE buffer (1 x TBE: 89 mM Tris; 89 mM boric acid; 2 mM EDTA; pH 8.3) for 2-3 h at 100 V and made visible by ethidium bromide staining and UV transillumination.

3. Results and discussion

Twenty-six reference strains of bifidobacteria were characterized by DNA restriction patterns using low-frequency cleavage endonucleases and PFGE. Two selected restriction enzymes, X&I and SpeI, were found to produce few genomic fragments which could be separated using PFGE. The sizes and numbers of fragments generated by digestion of intact DNA of bifidobacteria strains are listed in Table 3. Lambda concatemers and S. cereuisiue chromosomes were used as markers to estimate the size of fragments. Table 3 shows that strain-specific discrimination of bifidobacteria may be obtained following digestion of genomic DNA with XbaI and SpeI. According to the number of fragments generated, four different genomic fingerprints were discernible for B. animalis strains (ATCC 27673: 12, Type strain ATCC 25527: 13, ATCC 27672: 14, and ATCC 27536: 14 XZxzI fragments, respectively; ATCC 27673: 10, ATCC 27672: 12, ATCC 27536: 13, Type strain ATCC 25527: 16 SpeI fragments, respectively). Five different profiles could be observed for B. bifidum (Type strain ATCC 29521: 9, ATCC 11863: 9, ATCC 15696: 11, DSM 20082: 11, DSM 20215: 14 XbuI fragments, respectively; Type strain ATCC 29521: 12, DSM 20215: 12, ATCC 15696: 12, DSM 20082: 13, ATCC 11863: 16 S’eI fragments, respectively). Three genomic fingerprints were obtained for B. breve (Type strain ATCC 15700: 16, ATCC 15698: 17, ATCC 15701: 19 XbuI fragments, respectively; ATCC 15698: 17, Type strain ATCC 15700: 19, ATCC 15701: 19 SpeI fragments, respectively). Five genomic profiles were found for B. infuntis (ATCC 15702: 16, ATCC 27962: 17, ATCC 27920: 17, ATCC 17930: 18, Type strain ATCC 15697: 24 XbuI fragments, respectively; ATCC 15702: 14, ATCC 17930: 18, Type strain ATCC 15697: 18, ATCC 25962: 19, ATCC 27920: 22 SpeI fragments, respectively). Finally, three molecular types could be differentiated for reference strains of B. longum based on restriction fragments (Type strain ATCC 15707: 16, ATCC 15708: 17, DSM 20097: 18 XbuI fragments, respectively; Type strain ATCC 15707: 16, ATCC 15708: 16, DSM 20097: 16 SpeI fragments, respectively).

B. infuntis and B. longum are difficult to distinguish using phenotypic characterization and DNA-DNA hybridization (Bahaka et al., 1993). Molecular typing of B. infuntis and B. longum may be obtained using PFGE. Table 3 indicated that genomic fingerprints of reference strains of B. infuntis are different from those of B. longum. These data suggest that strains of B. infuntis could be separated from those of B. Zongum.

The molecular size of the genomic DNA was determined by adding the size of all the restriction fragments generated by XbuI and SpeI. Band sizes and the estimation of genome sizes for bifidobacterial species are summarized in Table 3. The B. animalis genome size varied between 1.2 and 1.5 Mb, depending on the

Tab

le

3 G

enom

ic

rest

rict

ion

an

alys

is o

f re

fere

nce

st

rain

s of

B

ifid

obac

teri

um

by p

uls

ed-f

ield

ge

l el

ectr

oph

ores

is.

Str

ain

E

nzy

me

Tot

al n

um

ber

Siz

e (K

b) o

f fr

agm

ents

of

res

tric

ted

frag

men

ts

Bif

idob

acte

rium

ani

rnal

is

DS

M 2

0104

X

bal

Spe

J

AT

CC

25

521

Xba

I Sp

el

AT

CC

21

614

Xba

J Sp

el

AT

CC

27

673

Xba

I S

peJ

AT

CC

21

612

BaJ

Sp

e I

AT

CC

21

536

Xba

I Sp

eI

13

16

13

16

13

16

12

10

14

12

14

13

215,

187

, 15

4, 1

21,

105,

95,

86,

46,

36,

35,

33,

27,

25

238,

170

, 16

2, 1

15,

100,

95,

91,

86,

82,

75,

65,

56,

51,

46,

42,

28

275,

181

, 15

4, 1

21,

105,

95,

86,

46,

36,

35,

33,

21,

25

238,

170

, 16

2, 1

15,

100,

95,

91,

86,

82,

75,

65,

56,

51,

46,

42,

28

215,

187

, 15

4, 1

21,

105,

95,

86,

46,

36,

35,

33,

27,

25

238,

170

, 16

2, 1

15,

100,

95,

91,

86,

82,

75,

65,

56,

51,

46,

42,

28

227,

196

, 14

7, 1

15, 8

6, 1

1, 6

5, 6

2, 5

3,44

, 40

, 27

28

9, 2

38,

206,

133

, 12

1, 1

10,

100,

68,

40,

31

335,

289

, 15

4, 1

15,

105,

91,

82,

78,

62,

51,

44,

40,

33,

21

422,

196

, 16

2, 1

05, 9

1, 7

5, 5

9, 4

8, 4

4, 3

6, 3

1, 3

0 22

7, 1

78,

162,

127

, 95,

86,

71,

68,

62,

51,

40.

36,

26,

20

238,

196

, 19

0, 1

54,

133,

100

, 91,

65,

44,

36,

33,

30,

25

Gen

ome

size

(M

b)

1.2

1.5

1.2

1.5

1.2

1.5

1.1

1.3

1.5

1.3

1.2

1.3

Bif

idob

acte

rium

bif

idum

D

SM

202

15

Xba

I Sp

eI

DS

M 2

0082

X

baI

Spe

1 D

SM

204

56

Xba

I S

peJ

AT

CC

29

521

Xba

J S

peJ

AT

CC

15

696

Xba

I S

peI

AT

CC

11

863

Xba

I S

peI

14

625,

405

, 24

0, 1

78,

165,

135

, 91,

84,

71,

63,

61,

44,

38,

34

2.2

12

240,

146

, 14

0, 1

35,

120,

94,

91,

84,

69,

43,

38

1.3

11

598,

325

, 11

8, 1

65,

106,

102

, 80,

71,

61,

43,

32

1.8

13

387,

325

, 26

1, 2

40,

193,

135

, 94,

91,

84,

77,

61,

43,

38

2.1

9 42

3, 3

71,

355,

178

, 16

5, 1

11, 9

1, 8

7, 3

6 1.

8 12

38

7, 2

98,

171,

146

, 13

5, 1

06,

102,

87,

84,

43,

38,

35

1.6

9 42

3, 3

71,

355,

178

, 16

5, 1

11. 9

1, 8

7, 3

6 1.

8 12

38

7,29

8,

171,

146

, 13

5, 1

06,

102,

87,

84,

43,

38,

35

1.6

11

355,

273,

17

8, 1

71,

146,

125

, 10

2, 7

7,69

,61,

36

1.8

12

387,

298

, 17

8, 1

58,

146,

125

, 12

0, 9

1, 8

4, 7

7, 4

3, 3

8 1.

7 9

355,

325

, 17

1, 1

58,

152,

146

, 98,

87,

69

1.6

16

325,

185

, 15

8, 1

46,

140,

135

, 13

0, 1

20,

102,

98,

87,

84,

80,

77,

43,

38

1.7

Bif

idob

acte

n’um

bre

ve

DS

M 2

0091

X

baI

SpeI

A

TC

C

1569

8 X

baI

SpeI

A

TC

C

1570

0 X

baI

Spe

I

476,

331

, 26

8, 1

50,

115,

96,

71,

65,

57,

54,

50,

46,

40,

38,

35,

31,

29

348,

143

, 13

7, 1

20, 8

8, 8

5, 8

1, 6

8, 6

2, 5

2, 4

4, 4

2, 3

8, 3

2, 2

8, 2

2, 2

0 47

6,33

1,

268,

150

, 11

5, 9

6,71

, 65

, 57

, 54

, 50

,46,

40,

38,

35,

31,

29

348,

143

, 13

7, 1

20, 8

8, 8

5, 8

1, 6

8, 6

2, 5

2,44

, 42

, 38

, 32

, 28

, 22

, 20

47

6, 3

31,

268,

126

, 85,

81,

68,

59,

54,

52,

46,

40,

35,

34,

31,

18

348,

179

, 15

0, 1

31,

105,

88,

85,

77,

74,

65,

52,

46,

44,

38, 3

2, 2

8, 2

2, 2

0,

18

331,

171

, 13

7, 1

20,

110,

92,

81,

71,

68,

59,

54,

48,

44,4

0,

37,

32,2

7,2l

, 18

36

6, 2

68,

178,

171

, 14

3, 1

26,

110,

88,

85,

71,

68,

59,

52,4

8,

42,

38,

32,

28,

21

1.9

1.4

1.9

1.4

1.6

1.6

17

17

17

17

16

19

AT

CC

15

701

Xba

I 19

1.

6 P

2.

0 S

peI

19

Bif

idob

acte

rium

inf

anti

s D

SM

200

88

Xba

I 24

1.

7

SpeI

18

X

baI

24

1.4

1.7

SpeI

X

baI

SpeI

18

17

22

178,

164

, 13

4, 1

29,

109,

101

, 93,

86,

82,

76,

67,

60,

57,

55,

49,

45,

43,

40,

37,

34,3

0,

29,

27,2

4 26

1, 1

51,

119,

109

, 97,

93,

86,

76,

70,

65,

55,

43,

40,

37,

32,

30,

23,

20

178,

164

, 13

4, 1

29,

109,

101

, 93,

86,

82,

76,

67,

60,

57,5

5,

49,

45,

43,

40,

37,

34,

30,

29,

27,2

4 26

1, 1

51,

119,

109

, 97,

93,

86,

76,

70,

65,

55,

43,

40,

37,

32,

30,

23,

20

178,

151

, 11

9, 1

09, 8

9, 8

2, 7

0, 6

7, 6

5, 5

7, 5

3, 4

5, 4

1, 4

0, 3

7, 3

2, 3

0 12

3, 1

19,

105,

101

,93,

79

, 76

, 73

,67,

62

, 60

, 55

, 53

, 49

, 47

, 45

, 41

, 40

, 34

,32

30,2

7 28

7, 1

23,

119,

109

, 10

1, 9

7, 8

9, 8

2, 7

0, 6

7, 6

0,55

, 51

, 47

, 55

, 40

,35

344,

139

, 12

3, 1

05,

101,

79,

73,

67,

65,

62,

49,

47,

45,

41,

40,

35,

34,

30,

27

1.4

1.3

1.4

Xba

I S

peI

17

19

1.5

1.5

Xba

I 18

17

8, 1

29,

114,

101

, 93,

86,

67,

62,

60,

55,

51,

47,4

3,

37,

34,

28,2

5,24

1.

2 S

peI

18

129,

105

, 10

1, 9

3,86

, 79

, 73

, 67

, 62

,57,

51

, 47

, 45

, 41

, 35

, 32

, 30

, 25

1.

2 X

baI

16

145,

129

, 12

3, 1

09, 8

9, 8

2, 7

6, 7

0, 6

5, 6

0, 5

7, 4

5, 4

1, 4

0, 3

7,32

1.

2 S

peI

14

314,

129

, 11

4, 1

01, 8

6, 8

2, 7

3, 6

2, 5

5, 5

1,45

, 41

, 34

, 30

1.

2

AT

CC

15

697

AT

CC

27

920

AT

CC

25

962

AT

CC

17

930

AT

CC

15

702

Tab

le 3

(co

nti

nu

ed)

Str

ain

E

nzy

me

Tot

al n

um

ber

of r

estr

icte

d fr

agm

ents

Siz

e (K

b) o

f fr

agm

ents

G

enom

e si

ze (

Mb)

Bif

idob

acte

rium

lon

gum

D

SM

200

97

Xba

I

DS

M 2

0219

AT

CC

15

707

AT

CC

15

708

SpeI

16

20

0, 1

84,

111,

107

, 98,

86,

76.

70,

67,

62,

52,

48,

42,

37,

34,

31

1.3

Xba

I 16

16

9, 1

56,

143,

121

, 11

1, 1

07, 9

8, 8

6, 7

6, 6

7, 6

2,52

, 42

, 39

, 33

, 22

1.

4 Sp

eI

16

292,

200

, 14

9, 1

07, 9

4, 7

9, 7

6, 7

0, 5

9, 5

7, 5

0, 4

8, 4

0, 3

7, 3

3, 2

9 1.

4 X

bal

16

169,

156

, 14

3, 1

21,

11,

107,

98,

86,

76,

67,

62,

52,

42,

39,

33,

22

1.4

SpeI

16

29

2, 2

00,

149,

107

, 94,

79,

76,

70,

59,

57,

50,4

8,40

, 37

, 33

, 29

1.

4 X

baI

17

156,

149

, 13

1, 1

21,

107,

90,

79,

76,

67,

62.

59,

54,

44,

42,

39,

36,

31

1.3

Spe

I 16

12

1, 1

11,

102,

98,

86,

73,

70,

62,

57,

54,

52,

46,

42,

37,

34,

31

1.1

18

237,

192

, 14

3, 1

31,

121,

111

, 10

2, 9

8, 8

3, 7

3, 6

4, 5

2, 5

0, 4

4, 4

0, 3

4, 3

1,

1.6

22

D. Roy et al. /ht. J. Food Microbiology 29 (I 996) I I-29 21

242.5

194.0

145.5

97.0

48.5

242.5

194.0

145.5

97.0

48.5

Fig. 2. PFGE patterns of genomic DNA from commercial strains of B. animalis after digestion by (A)

XbaI and (B) SpeI.

restriction enzyme used. The B. bifdum strains exhibited the highest genome size, varying between 1.3 and 2.2 Mb. The genome sizes of B. breve, B. infantis and B. longum were estimated to be between 1.1 and 2.0 Mb, intermediate between those of B. animalis and B. bifidum. No fragments were considered as doublets due to their intensity and thus were not taken into account twice to estimate the genomic size obtained. Overall, the size estimates for the bifidobacterial species studied are comparable with the sizes already determined for other lactic acid bacteria (Tanskanen et al., 1990; Daniel et al., 1993; Roussel et al., 1993). These values are in agreement with the B. breve genome size of 2.1 Mb estimated by Bourget et al. (1993).

Standard commercially-available industrial strains of bifidobacteria were previously identified according to phenotypic characterization (Table 1). AI1 strains phenotypically identified as B. longum possessed N-acetyl+glucosaminidase activity and were melezitose- and xylose-positive. Strains of B. animalis could be differentiated from strains of B. longum by the presence of phosphohydrolase and P-glucosidase activities (data not shown). The commercial strains of B. longum were isolated from Japanese commercial preparations. These results indicated that phenotypic characteristics proposed by Yaeshima et al. (1992) and Roy et al. (1994) are useful for differentiation of commercially-available industrial strains of B. longum and B. animalis.

Fig. 2 indicates that PFGE profiles of all 11 commercial strains of B. animalis (principally isolated from European commercial preparations) were identical to B. animalis ATCC 27536, previously isolated from chicken feces. These results are in agreement with those of Biavati et al. (1992) who observed that B. animalis was

22 D. Roy et al. /hi. J. Food Microbiology 29 (1996) I1 -29

the only species present in the fermented milk preparations examined. These authors noted that the electrophoretic patterns of cellular proteins revealed identical bands for all the strains from the fermented milk products and the reference strain of B. animalis ATCC 27536. Roy et al. (1994) also found that commercially-available strains of B. animal& possessed P-galactosidase electrophoretic patterns identical to those of the reference strains of B. animalis. However, molecular typing using PFGE allowed definitive identification of these strains.

Seven discriminant electrophoretic patterns were found for B. longum strains (Fig. 3). The reference strains DSM 20097, ATCC 15707 and 15708 displayed specific PFGE patterns. Only one commercial strain of B. longum (RW-002) was identical to the type strain ATCC 15707. The other 14 commercially-available strains of B. longum (mainly isolated from Japanese commercial preparations) were divided into four new molecular types based on their PFGE patterns. Four strains were identical to RW-009, two strains shared identical PFGE profile with RW-008 and three strains exhibited a PFGE profile identical to RW-001. The commercial strain RW-020 possessed a unique PFGE pattern. The patterns were different for each molecular type, although some common bands were observed.

Reuter (1963) reported that B. longum strains were divided into two biovars a and b. Recently, it was recognized that B. longum strains, which are incapable of fermenting melezitose, can be detected by DNA-DNA hybridization (Yaeshima et al., 1991). Yaeshima et al. (1992) detected bifidobacterial strains from human feces which were identified as B. longum by DNA-DNA homology, but found to be distinct from typical strains of B. longum in fermentation pattern. These strains were divided into six phenotypic groups by the ability to ferment mannose, melezitose, mannitol, sorbitol and glucosides. In the current study, the use of PFGE allowed molecular typing of commercially-available industrial strains of B. longum. This species could be divided into seven molecular types based on genomic fingerprints of strains following digestion of genomic DNA with X&z1 or SpeI. PFGE is thus a reliable and practical method for comparing commercial strains of bifidobacteria. This method may also be used to determine the specific origin of strains, as observed for B. animalis.

Lauer and Kandler (1983) observed that B. breve and B. infuntis are genetically related to each other at a DNA-DNA homology level of 50%. In addition, B. longum is genetically closely related to B. infuntis (about 65% DNA-DNA homology). Our results indicate that strain differentiation within a particular species may be determined by PFGE. This method allowed molecular typing of B. breve, B. infuntis and B. longum. However, PFGE patterns do not allow speciation of bifidobacteria. Other methods must be developed for identification of commercially-important strains of Bifidobacterium at the species level.

Recently, Bahaka et al. (1993) indicated that B. infuntis and B. longum strains, including the type strains, were not differentiated phenotypically, even on the basis of a large number of tests (carbohydrate fermentation patterns and enzymatic activity profiles). These authors observed that 19 human strains showed 67 to 98% and 61 to 80% of DNA-DNA relatedness of B. longum and B. infantis, respec-

A Kb

228 242.5 194.0

145.5

97.0

48.5

B Kb

%9:8 242.5 194.0

145.5

97.0

48.5


mQ)CDcOrrhlr-0 r OCVNOcw(Uhl(\1 4 44444444 3 33333333 fY rfLYu:ctfElYlYcc

Fig. 3. PFGE patterns of genomic DNA from commercial strains of B. Zongum after digestion by (A)

XbaI and (B) SpeI.

tively. They concluded that B. longum and B. infantis could not be differentiated on the basis of a large number of phenotypic characteristics and the genetic data lead them to question the existence of strains named B. infantis.

PCR amplification based on specific DNA-primers from 16s rDNA of bifidobacteria (Table 2) was used to reliably identify commercial strains of B. longum. Fig. 4 and Table 4 show that the primers designed for B. infuntis (InfUS-InfL6) yielded PCR products of 565 bp (Table 2) with the reference strains of B. infantis

24

Table 4

D. Roy et al. / Int. .I. Food Microbiology 29 (1996) 11-29

Strains tested for the oresence of target secmences bv PCR

Species Strains Primers Primers

BreU3-BreL4 InfU5-Infl6

Primers

LonU7-LonL8

B. bifidum

B. breve

B. infantis

B. longum

B. animalis B. magnum B. indicum B. suis B. thermophilum Lb. acidophilus Lb. casei

ATCC 15696

ATCC 29521 ATCC 11863

S 28-a a RW-012

DSM 20215

DSM 20082

DSM 20456

ATCC 15698

ATCC 15700

ATCC 15701

s-17c a

S-46 a

RW-010

DSM 20091 ATCC 15697

ATCC 25962

ATCC 27920 G

ATCC 15702

ATCC 17930

DSM 20088

ATCC 15707

ATCC 15708 DSM 20097

DSM 20219

RW-001

RW-002

RW-008

RW-009

RW-019

RW-020

RW-021

RW-022

RW-023

RW-024

RW-025

RW-026

RW-027 RW-028

ATCC 25527 ATCC 27540 ATCC 25912 ATCC 27533

ATCC 25525 ATCC 4356 ATCC 393

- -

- - - -

- _ _

_

- - _ _

- - -

-

_ _ + + + + + + + + + + + + + + + + + + _ + _ + +

D. Roy et al. / Int. J. Food Microbiology 29 (I 996) I I-29 25

Table 4 (continued)

Species Strains Primers Primers BreU3-BreL4 InfUS-Inf16

Primers

LonU7LonL8

Lb. rhamnosus ATCC 7469 _ _ -

Lb. bulgaticus EYE-41Lb - - _

0129 b - - _

Srr. thermophilus ATCC 19258 _ _ _

Lc. lactis subsp. lactis CNRZ 1075 = - _ -

a Strains supplied by G. Reuter, Freie Universitat, Berlin, Germany.

b Strains isolated from commercial yogurts. ’ Strain obtained from CNRZ collection, Jouy-En-Josas, Institut National de la recherche agronomique,

France.

(except B. infantis DSM 20088). Only B. indicum ATCC 25912 also gave positive results with these primers (Table 4). Fig. 4 shows that the primers specific for B. breve (BreU3-BreL4) yielded a PCR product of 811 bp (Table 2) with B.

ABCDE FGH I JK LMNOPQRSTUVWX

bp

1353 1078

872

603

310

Fig. 4. Typical gel obtained after electrophoresis of PCR-amplified DNA with the Bifidobacterium- specific primers described in Table 3. Lanes A and X, 4X174/Hae III markers (Promega Corporation,

Madison, WI, USA). Primers InfU5 and InfL6, Lane: B, B. infantis ATCC 15696; C, B. infantis ATCC 25962; D, B. infantis ATCC 27920; E, B. infantis ATCC 15707; F, B. infantis ATCC 17930; G, B. infantis DSM 20088. Primers BreU3 and BreL4, Lane: H, B. breve ATCC 15698; I, B. breve ATCC 15700; J, B. breve ATCC 15701; K, B. breoe ~-17~; L, B. breve s-46, M, B. breve RW-010, N, B. brew DSM 20001. Primers LonU7 and LonL8, Lane: 0, B. longum ATCC 15707; P, B. longum ATCC 15708;

Q, B. longum DSM 20097; R, B. longum RW-001; S, B. longum RW-002; B. longum RW-008; B. longum RW-009; B. longum RW-020; W, negative control.

26 D. Roy et al. /ht. J. Food Microbiology 29 (I 996) II-29

strains (except with B. brece ATCC 15698). The other strains of bifidobacteria (non-B. breve) were all negative (Table 4). All strains of B. longum (reference and commercial strains) gave PCR products of 567 bp (Table 2) by using the specific primers for B. Zongum (Table 4). Among other strains of bifidobacteria and lactic acid bacteria, PCR products were also obtained with B. magnum, B. suis and B. thermophilum (Table 4).

Yamamoto et al. (1992) also observed that probes for bifidobacteria of human origin cross-reacted with a few strains of heterologous Bifidobacterium species of non-human origin. However, their probes were highly species specific against strains of human origin, which is in agreement with our results. In addition, Yamamoto et al. (1992) noted that although there was only one base difference between B. infuntis and B. longum at the target site for their respective probes, it

Aabababababababababababab A

Fig. 5. PCR amplification of DNA extracted from strains of Bifidobacferium by using specific primers. a: primers LonU7 and LonL8; b: primers InfU5 and InfL6. A: bX174/Hae III markers.

Tab

le

5

Phen

otyp

ic

char

acte

rist

ics

of

stra

ins

of

hum

an

orig

in

test

ed

for

the

pres

ence

of

ta

rget

se

quen

ces

by

PCR

Cha

ract

eris

tics

89-1

71

a8Y

-172

89-1

7489

-177

89-1

8689

-193

89-2

1689

-239

89-2

4589

-247

89-2

5789

-259

89-2

6O89

-263

89-2

67

89-2

6889

-276

89-2

8189

-287

89-2

90

Eem

ymat

ic

test

s

Alk

alin

e ph

osph

atas

e -

Cys

tine

amin

opep

- -

tidas

e

Phos

phoh

ydro

lase

-

p -

Gal

acto

sida

se

+

/3 -

Glu

cosi

dase

+

N-A

cety

l-/3

-glu

cos-

+

amin

idas

e

Fer

men

tati

on

patt

erns

L - A

rabi

nose

+

Rib

ose

+

D-X

ylos

e +

o-M

anno

se

+

Lac

tose

+

Mel

ibio

se

+

Cel

lobi

ose

_

Mel

ezito

se

_

Raf

fino

se

+

Gly

coge

n _

Man

nito

l -

Salic

in

_

PC

R

test

s

_ +

_ +

_ + +

+

+

+ +

+

+

+ +

_ - _

Prim

ers

Bre

U3-

Bre

L4V

D

” N

D

Prim

ers

InfU

S-In

fL6

- -

Prim

ers

Lon

U7-

Lon

LB

+

+ +

_ +

+

_ _ _ + +

+

+ - _ +

_ - +

- _ _

_ _ _ +

_ - +

- _ _ +

+

- + +

_ _ _ ND

_ +

- - _ + _ +

+ _ +

+

+ +

- +

+

_ _ - ND

- +

- +

_ + - +

+ - +

+ +

+

+

+

+

_ _ _ ND

_ +

_ _ _ + _ +

_ - +

_ +

+

_ +

+ _ - - ND

- +

- - _ +

_ + +

+

+

_ +

+

+

+

+ - _ _ ND

_ +

_ _ _ +

_ +

+

+

+ +

+

+

+

+

+

_ _ _ ND

_ +

- - _ +

- +

+ _ +

_ + +

_ +

+ - _ - ND

- +

_ _ _ + - - - _ +

- +

+

- +

+ - _ _ ND

_ +

_ _ - +

_ _ _ _ _ - + +

+

+

+

_ _ _ ND

_ +

- _ _ + _ _ +

_ +

_ + +

_ +

+

_ _ - ND

- +

_ _ - +

_ +

+

_ +

- +

+

- +

+

_ _ _ ND

_ +

+

- _ +

_ + +

- +

_ +

+

_ +

+ _ _ - ND

- +

+

_ - +

_ +

+ _ +

- +

+ - +

+ _ _ _ ND

_ +

_ - _ +

_ +

+

+

+

_ +

+

_ + +

_ _ - ND

- +

_ _ _ +

- +

+

_ +

- + +

_ - + _ - _ ND

_ +

_ _ - +

_ +

+

_ +

_ +

+

_ +

+

- _ _ ND

_ +

-

a St

rain

s of

hu

man

or

igin

is

olat

ed

by

Bah

aka

et

al.

(199

3).

b N

D

= no

t de

term

ined

.

_ _ ND

- +

Y

28 D. Roy et al. /ht. J. Food Microbiology 29 (1996) 1 I-29

was possible to differentiate B. longurn from B. infantis. Our results also indicate that B. longum could be differentiated from B. infantis although there were only three different bases between the respective primers of these two species (different base-pairs were at positions 996 and 1003 in L.onU7 and InfUS, and position 461 in

LonLS and InfL6; Fig. 1). Twenty human strains isolated from child feces and adult intestine by Bahaka et

al. (1993) (Table 1) were characterized phenotypically and examined by using the primers designed for B. breve, B. infantis and B. Zongum (Table 5). According to Bahaka et al. (1993), the DNA-DNA relatedness values of these isolates were very simiIar to those of type strains of B. Lungum and B. infantis. Only one isolate (89-174) gave negative results with specific DNA-primers for B. breve, B. infantis and B. longum (Table 5 and Fig. 5), which was identified as B. adolescentis according to numerical analysis of phenotypic characters (Table 1). Nineteen strains were identified as B. longum according to our numerical analysis of phenotypic characters (Table 1). Most of them were N-acetyl+glucosaminidase and meIezitose-positive, and P-glucosidase-negative (Table 5). The use of PCR confirmed the identification of these 19 isolates, since only the primers specific for B. longum yielded PCR products (Fig. 5 and Table 5). Our results support the proposition that B. infantis and B. fongum are distinct species (Frothingham et al., 1993). We have shown that PCR amplification using DNA primers derived from 16s rDNA provides a highly specific technique for distinguishing strains of B. brer)e, B. infantis and B. longum.

Acknowledgements

We thank Drs. F. Gavini, R. McKellar and G. Reuter for supplying the strains used in this study.

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