Identification of species belonging to the Bifidobacterium genus by PCR-RFLP analysis of a hsp60 gene fragment.

BACKGROUND: Bifidobacterium represents one of the largest genus within the Actinobacteria, and includes at present 32 species. These species share a high sequence homology of 16S rDNA and several molecular techniques already applied to discriminate among them give ambiguous results.
RESULTS: This work illustrates a simple and cheap molecular tool for the identification of Bifidobacterium species. The hsp60 universal primers were used in a simple PCR procedure for the direct amplification of 590 bp of the hsp60 sequence. The in silico restriction analysis of bifidobacterial hsp60 partial sequences allowed the identification of a single endonuclease (HaeIII) able to provide different PCR-restriction fragment length polymorphism (RFLP) patterns in the Bifidobacterium spp. type strains evaluated. The electrophoretic analyses allowed to confirm the different RFLP patterns.
CONCLUSIONS: The developed PCR-RFLP technique resulted in efficient discrimination of the tested species and subspecies and allowed the construction of a dichotomous key in order to differentiate the most widely distributed Bifidobacterium species as well as the subspecies belonging to B. pseudolongum and B. animalis.

Background

Members of the genus Bifidobacterium are Gram-positive, obligate anaerobic, non-motile, non-spore forming bacteria [1], and are the most important constituents of human and animal intestinal microbiota [2,3]. Recently, news species of bifidobacteria have been described [4-6] and now more than 30 species have been included in this genus.

Bifidobacterium spp. can be detected in various ecological environments, such as intestines of different vertebrates and invertebrates, dairy products, dental caries and sewage. Considering the increasing application of Bifidobacterium spp. as protective and probiotic cultures [7-9], and the fast enlargement of the genus, easy identification tools to discriminate new isolates are essential. Moreover, their correct taxonomic identification is of outmost importance for their use as probiotics [2]. Conventional identification and classification of Bifidobacterium species have been based on phenotypic and biochemical features, such as cell morphology, carbohydrate fermentation profiles, and polyacrylamide gel electrophoresis analysis of soluble cellular proteins [10]. In the last years several molecular techniques have been proposed in order to identify bifidobacteria. Most available bifidobacterial identification tools are based on 16S rRNA gene sequence analysis, such as ARDRA [11,12], DGGE [13] and PCR with the use of species-specific primers [14-16]. However, 16S rDNA of Bifidobacterium spp. has a high similarity, ranging from 87.7 to 99.5% and bifidobacterial closely related species (e.g. B. catenulatum and B. pseudocatenulatum) or subspecies (e.g. B. longum and B. animalis subspecies) even possess identical 16S rRNA gene sequences [17,18]. For this reason different molecular approaches have been tested based on repetitive genome sequences amplification, such as ERIC-PCR [19,20], BOX-PCR [21,22] or RAPD fingerprinting analysis [23]. These fingerprinting methods have the disadvantage of a low reproducibility, and they need strict standardization of PCR conditions. The use of different polymerases, DNA/primer ratios or different annealing temperatures may lead to a discrepancy in the results obtained in different laboratories [24].

In recent years alternative molecular markers have been proposed for bifidobacteria identification (e.g. hsp60, recA, tuf, atpD, dnaK) and Ventura et al. [18] developed a multilocus approach, based on sequencing results, for the analysis of bifidobacteria evolution. The hsp60 gene, coding for a highly conserved 60 kDa heat-shock-protein (a chaperonin), has been evaluated for phylogenetic analysis in bifidobacteria by Jian et al. [25]. The sequence comparison of this gene has been already used for species identification and phylogenetic analysis of other genera (e.g. Staphylococcus, Lactobacillus) and enteric pathogens [26-28]. A chaperonin database (cpnDB) is available on line, collecting bacterial and eukaryotic sequences (http://www.cpndb.ca/cpnDB/home.php) [29].

The purpose of this study is the development of a rapid, reproducible and easy-to-handle molecular tool for the identification of Bifidobacterium species isolated from various environments. The protocol is based on the restriction endonuclease analysis of the PCR-amplified hsp60 partial gene sequence (hsp60 PCR-RFLP) with the use of a single restriction enzyme and has been tested on the 30 most widely distributed Bifidobacterium species and subspecies. A diagnostic dichotomous key to speed up profile interpretation has also been proposed.

Methods

Bacterial strains and culture conditions

The type strains used to develop the technique are listed in Table  1, whereas the strains used to validate the method are reported in Table  2. The strains, belonging to BUSCoB (Bologna University Scardovi Collection of Bifidobacteria) collection, were isolated from faeces of human and animals and from sewage. Bacteria were maintained as frozen stocks at −80°C in the presence of skim milk as cryoprotective agent. Working cultures were prepared in TPY medium [1], grown anaerobically at 37°C and harvested at logarithmic phase.

Table 1

Type-strains investigated

Species	International culture collection
Bifidobacterium adolescentis	ATCC 15703
Bifidobacterium angulatum	ATCC 27535
Bifidobacterium animalis subsp. animalis	ATCC 25527
Bifidobacterium animalis subsp. lactis	DSM 10140
Bifidobacterium asteroides	ATCC 25910
Bifidobacterium bifidum	ATCC 29521
Bifidobacterium boum	ATCC 27917
Bifidobacterium breve	ATCC 15700
Bifidobacterium catenulatum	ATCC 27539
Bifidobacterium choerinum	ATCC 27686
Bifidobacterium coryneforme	ATCC 25911
Bifidobacterium cuniculi	ATCC 27916
Bifidobacterium dentium	ATCC 27534
Bifidobacterium gallicum	ATCC 49850
Bifidobacterium gallinarum	ATCC 33777
Bifidobacterium indicum	ATCC 25912
Bifidobacterium longum subsp. longum	ATCC 15707
Bifidobacterium longum subsp. infantis	ATCC 15697
Bifidobacterium longum subsp. suis	ATCC 27533
Bifidobacterium minimum	ATCC 27539
Bifidobacterium merycicum	ATCC 49391
Bifidobacterium pseudolongum subsp pseudolongum	ATCC 25526
Bifidobacterium pseudolongum subsp. globosum	ATCC 25865
Bifidobacterium pseudocatenulatum	ATCC 27919
Bifidobacterium pullorum	ATCC 27685
Bifidobacterium ruminantium	ATCC 49390
Bifidobacterium subtile	ATCC 27537
Bifidobacterium thermacidophilum subsp. porcinum	LMG 21689
Bifidobacterium thermacidophilum subsp. thermacidophilum	LMG 21395
Bifidobacterium thermophilum	ATCC 25525

Table 2

List of strains investigated to confirm the conservation of RFLP profiles (strains belonging to BUSCoB collection)

Species*	Strain	Source
Bifidobacterium animalis subsp. animalis	T169	Rat
Bifidobacterium animalis subsp. animalis	T6/1	Rat
Bifidobacterium animalis subsp. lactis	P23	Chicken
Bifidobacterium animalis subsp. lactis	F439	Sewage
Bifidobacterium animalis subsp. lactis	Ra20	Rabbit
Bifidobacterium animalis subsp. lactis	Ra18	Rabbit
Bifidobacterium animalis subsp. lactis	P32	Chicken
Bifidobacterium bifidum	B1764	Infant
Bifidobacterium bifidum	B2091	Infant
Bifidobacterium bifidum	B7613	Preterm infant
Bifidobacterium bifidum	B2009	Infant
Bifidobacterium bifidum	B2531	Infant
Bifidobacterium breve	B2274	Infant
Bifidobacterium breve	B2150	Infant
Bifidobacterium breve	B8279	Preterm infant
Bifidobacterium breve	B8179	Preterm infant
Bifidobacterium breve	Re1	Infant
Bifidobacterium catenulatum	B1955	Infant
Bifidobacterium catenulatum	B684	Adult
Bifidobacterium catenulatum	B2120	Infant
Bifidobacterium pseudocatenulatum	B1286	Infant
Bifidobacterium pseudocatenulatum	B7003
Bifidobacterium pseudocatenulatum	B8452
Bifidobacterium dentium	Chz7	Chimpanzee
Bifidobacterium dentium	Chz15	Chimpanzee
Bifidobacterium longum subsp.longum	PCB133	Adult
Bifidobacterium longum subsp. infantis	B7740	Preterm infant
Bifidobacterium longum subsp. infantis	B7710	Preterm infant
Bifidobacterium longum subsp. suis	Su864	Piglet
Bifidobacterium longum subsp. suis	Su932	Piglet
Bifidobacterium longum subsp. suis	Su905	Piglet
Bifidobacterium longum subsp. suis	Su908	Piglet
Bifidobacterium pseudolongum subsp. pseudolongum	MB9	Chicken
Bifidobacterium pseudolongum subsp. pseudolongum	MB10	Mouse
Bifidobacterium pseudolongum subsp. pseudolongum	MB8	Chicken
Bifidobacterium pseudolongum subsp. globosum	Ra27	Rabbit
Bifidobacterium pseudolongum subsp. globosum	VT366	Calf
Bifidobacterium pseudolongum subsp. globosum	T19	Rat
Bifidobacterium pseudolongum subsp. globosum	P113	Chicken

* previously assigned taxonomic identification.

In silico analysis

An in silico analysis was performed for the evaluation of a suitable restriction enzyme. Available hsp60 sequences had been retrieved from cpnDB database and GeneBank, thanks to the work of Jian et al. [25]. In silico digestion analysis was carried out on fragments amplified by universal primers H60F-H60R [30] using two on-line free software: webcutter 2.0 (http://rna.lundberg.gu.se/cutter2) and http://insilico.ehu.es/restriction softwares [31]. Blunt end, frequent cutter enzymes that recognize not degenerated sequences have been considered in order to find a suitable enzyme for all the species (e.g. RsaI, HaeIII, AluI, AccII). However in silico analysis had been performed also on sticky end enzymes (e.g. AatII, Sau3AI, PvuI).

DNA extraction from pure cultures

10 ml of culture were harvested and washed twice with TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.6), resuspended in 1 ml TE containing 15 mg lysozyme and incubated at 37°C overnight. Cells were lysed with 3 ml of lysis buffer (100 mM Tris–HCl, 400 mM NaCl, 2 mM EDTA, pH 8.2), 220 μl SDS (10% w/v) and 150 μl proteinase K (>600 mAU/ml, solution) and incubated for 2 hours in water bath at 60°C. One ml of saturated NaCl solution was added and the suspension was gently inverted twice. Pellets were harvested through centrifugation (5000 × g) at room temperature for 15 minutes. After the transfer of clean supernatants in new tubes, DNA was precipitated with 2.5 volumes of cold ethanol (95%) and resuspended in 300 μl of TE buffer [32].

Amplification of gene hsp60 and restriction with HaeIII

Universal primers were used to amplify approximately 600 bp of the hsp60 gene in the Bifidobacterium spp. investigated. These primers H60F (5‘-GG(ATGC)GA(CT)GG(ATGC)AC(ATGC)AC(ATGC)AC(ATGC)GC(ATGC)AC(ATGC)GT-3’) and H60R (5’-TC(ATGC)CC(AG)AA(ATGC)CC(ATGC)GG(ATGC)GC(CT)TT(ATGC)AC(ATGC)GC-3’) were designed by Rusanganwa et al. [30] on the basis of the conserved protein sequences GDGTTATV and AVKAPGFGD in HSP60. Amplifications were performed in 20 μl volumes with 1.5 μM of each primer (Eurofins MWG Operon, Ebersberg, Germany), 10 μl 2X HotStarTaq Plus Master Mix (Qiagen, Italy) (1,5 mM MgCl2, 1 U Taq, 0.2 mM dNTP, final concentration) and 150 ng/μl DNA. The PCR cycle consisted of an initial denaturation of 5 min at 95°C followed by 35 cycles of denaturation (30s at 94°C), annealing (30s at 61°C) and extension (45 s at 72°C). The PCR was completed with a final elongation of 10 min at 72°C. The PCR amplification was performed with a PCR Verity 96-well thermal cycler (Applied Biosystems, Milan, Italy). After amplification, the product was visualized via agarose gel (1.3% w/v) in 1X TBE buffer and visualized with ethidium bromide under UV light. A 100 bp DNA ladder (Sigma-Aldrich) was used as a DNA molecular weight marker. Bands were excised from agarose gel (Additional file 1: Figure S1) and DNA was eluted with NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel GmbH & Co. KG, Germany) in order to avoid possible non-specific amplifications. 3 μl of the eluted DNA was re-amplified in a 30 μl PCR reaction (see above). BSA was added to the reaction (5% v/v, Fermentas). The PCR products (2 μl) were checked for non-specific amplification on agarose gel. 20 μl (~6 μg) of PCR amplicons were digested with HaeIII enzyme. Restriction digestion was carried out for 2 h at 37°C in 30 μl reaction mixture with 1X SM Restriction Buffer (Sigma-Aldrich), 1.5 μl HaeIII (10 U/μl, Sigma-Aldrich) and water. Digestion products were stained with ethidium bromide and visualized under UV-light (GelDoc™, BioRad), after agarose gel electrophoresis (3.0% agarose (w/v), TBE 1X) at 210 V (3 h). A 20 bp DNA ladder (Sigma-Aldrich) was used. The obtained pictures were elaborated with a free software GNU Image Manipulation Program (Gimp 2.8) only to invert colors and increase contrast.

Precast gradient polyacrylamide gels (4-20%) (Lonza Group Ltd, Switzerland) were also used to obtain RFLP profiles, in order to have a comparison with agarose gels. The vertical electrophoresis apparatus used was P8DS™ Emperor Penguin (Owl, Thermo Scientific) with an adaptor for Lonza precast gels. The run was performed at 100 V in TBE 1X.

Diagnostic key

A dichotomous key was developed comparing in silico digestion results and the evaluation of visible bands with the use of ImageLab™ 2.0 software (Bio-Rad Laboratories, Inc.).

Results and discussion

The analysis and comparison of restriction profiles obtained with in silico digestion of bifidobacterial hsp60 sequences allowed the identification of a set of appropriate frequent-cutter endonucleases that recognize non degenerated sequences. The restriction enzyme HaeIII was found to give the clearest and most discriminatory profiles in theoretical PCR-RFLP patterns, discriminating the majority of Bifidobacterium type-strains tested (Table  3). Furthermore, the profiles of other strains, belonging to the investigated species, have been analyzed to confirm the conservation of RFLP profiles within species.

Table 3

Expected fragment sizes obtained with digestion of the gene sequences

Bifidobacterium species	GenBank entry	Predicted fragment sizes	Profile
B. adolescentis	AF210319	31-36-81-103-339
B. angulatum	AF240568	42-54-59-139-296
B. animalis subsp. animalis	AY004273	17-53-86-97-114-223
B. animalis subsp. lactis	AY004282	71-86-96-114-223
B. asteroides	AF240570	30-38-75-97-109-242
B. bifidum	AY004280	22-31-59-181-297
B. boum	AY004285	22-117-200-251
B. breve	AF240566	106-139-139-200
B. catenulatum	AY004272	53-198-338
B. choerinum	AY013247	36-42-51-52-54-59-97-200
B. coryneforme	AY004275	16-32-54-158-338
B. cuniculi	AY004283	16-42-53-70-128-281
B. dentium	AF240572	22-31-42-68-130-139-158
B. gallicum	AF240575	42-253-297
B. gallinarum	AY004279	16-31-42-81-139-281
B. indicum	AF240574	16-32-36-42-45-123-296
B. longum subsp. longum	AF240578	42-113-138-139-158	*
B. longum subsp. infantis	AF240577	42-113-138-139-158	*
B. longum subsp. suis	AY013248	42-113-138-139-158	*
B. merycicum	AY004277	22-31-42-59-139-297
B. minimum	AY004284	16-51-60-66-70-327
B. pseudocatenulatum	AY004274	42-53-198-297
B. pseudolongum subsp pseudolongum	AY004282	17-22-30-32-42-42-109-297
B. pseudolongum subsp. globosum	AF286736	16-17-22-30-32-42-109-323
B. pullorum	AY004278	16-31-36-42-81-87-297
B. ruminantium	AF240571	31-106-114-339
B. subtile	Not available	Not avaiable	+
B. thermacidophilum subsp porcinum	AY004276	20-42-53-59-97-139-180	*†
B. thermacidophilum subsp thermacidophilum	AY004276	20-42-53-59-97-139-180	*†
B. thermophilum	AF240567	54-59-117-139-222

+ hsp60 sequence of B. subtile type strain was not available in the press-time.

† the available sequences at GeneBank and cpnDB belonged to B. thermacidophilum (with no distinction in subspecies).

*subspecies not discernable.

Amplification and restriction analysis of Bifidobacterium spp

Theoretical restriction profiles have been confirmed in vitro on agarose gel. The obtained fragments ranged from 16 bp to 339 bp (Table  3). Fragments lower than 25 bp were not considered as they did not help in species discrimination and in addition they co-migrate with primers. Time course analysis of restricted samples showed the formation of a band of ~200 bp in several species due to an over-digestion (data not shown) and this invalidated the RFLP profiles. For this reason the protocol has been optimized at 2 hours restriction time. Fragments greater than 360 bp were also not considered due to a possible incomplete digestion of such long fragments.

The obtained gels (Figures  1, 2, 3, 4 and 5) show species-specific profiles for all type-strains other than B. longum and B. thermacidophilum subspecies. This technique does not allow the identification of the subspecies belonging to these species, which displayed identical RFLP profiles. Matsuki et al. [14,17] proposed specific primers to differentiate the subspecies of the species B. longum, while B. thermacidophilum subsp. porcinum and B. thermacidophilum subsp. thermacidophilum can be differentiated according to Zhu et al. [33]. The proposed restriction analysis is efficient in discriminating very closely related species and subspecies as B. catenulatum/B. pseudocatenulatum, B. pseudolongum subsp. pseudolongum/B. pseudolongum subsp. globosum and B. animalis subsp. animalis/B. animalis. subsp. lactis.

Figure 1

Agarose gel electrophoresis of digested DNA fragments with HaeIII (negative image). Lane1, ladder 20 bp (Sigma-Aldrich); Lane 2, B. bifidum ATCC 29521; Lane 3, B. asteroides ATCC 25910, Lane 4, B. coryneforme ATCC 25911; Lane 5, B. indicum ATCC 25912; Lane 6, B. thermophilum ATCC 25525; Lane 7, B. boum ATCC 27917; Lane 8, B. thermacidophilum subsp. porcinum LMG 21689; Lane 9, B. thermacidophilum subsp. thermacidophilum LMG 21395; Lane 10, ladder 20 bp (Sigma-Aldrich).

Figure 2

Agarose gel electrophoresis of digested DNA fragments with HaeIII (negative image). Lane1, ladder 20 bp (Sigma-Aldrich); Lane 2, B. minimum ATCC 27539; Lane 3, B. pullorum ATCC 27685, Lane 4, B. subtile ATCC 27537; Lane 5, B. gallinarum ATCC 33777; Lane 6, ladder 20 bp (Sigma-Aldrich).

Figure 3

Agarose gel electrophoresis of digested DNA fragments with HaeIII (negative image). Lane1, ladder 20 bp (Sigma-Aldrich); Lane 2, B. breve ATCC 15700; Lane 3, B. longum subsp. infantis ATCC 15697; Lane 4, B. longum subsp. longum ATCC 15707; Lane 5, B. longum subsp. suis ATCC 27533; Lane 6, ladder 20 bp (Sigma-Aldrich).

Figure 4

Agarose gel electrophoresis of digested DNA fragments with HaeIII (negative image). Lane1, ladder 20 bp (Sigma-Aldrich); Lane 2, B. merycicum ATCC 49391; Lane 3, B. angulatum ATCC 27535, Lane 4, B. pseudocatenulatum ATCC 27919; Lane 5, B. catenulatum ATCC 27539; Lane 6, B. dentium ATCC 27534; Lane 7, B. ruminantium ATCC 49390; Lane 8, B. adolescentis ATCC 15703; Lane 9, ladder 20 bp (Sigma-Aldrich).

Figure 5

Agarose gel electrophoresis of digested DNA fragments with HaeIII (negative image). Lane1, ladder 20 bp (Sigma-Aldrich); Lane 2, B. gallicum ATCC 49850; Lane 3, B. choerinum ATCC 27686, Lane 4, B. animalis subsp. lactis DSM 10140; Lane 5, B. animalis subsp. animalis ATCC 25527; Lane 6, B. cuniculi ATCC 27916; Lane 7, B. pseudolongum subsp. pseudolongum ATCC 25526; Lane 8, B. pseudolongum subsp. globosum ATCC 25865; Lane 9, ladder 20 bp (Sigma-Aldrich).

The same method has been applied with the use of precast gradient polyacrylamide gels. The resolution was greater than that obtained on agarose gels, loading only 4 μl of the restriction reaction instead of the 30 μl used in horizontal electrophoresis. This may allow to reduce the volume of amplification reactions with a consequent reduction of costs.

The comparison between in silico digestion and the obtained gel profiles allowed to develop a dichotomous key (Figure  6) for a faster interpretation of the restriction profiles.

Figure 6

Dichotomous key to identify species of based upon HaeIII restriction digestion of ~590 bp of the gene.

Validation of PCR-RFLP analysis on bifidobacterial isolates

39 strains belonging to 12 different species/subspecies (Table  2) have been investigated to validate the PCR-RFLP technique. Most of the strains tested were previously identified using biochemical tests and in some cases also molecular techniques (species-specific PCR, 16S rDNA sequencing). The obtained data confirmed a conservation of the profiles concerning the species and subspecies tested. Two figures are available as Additional files (Additional file 2: Figure S2: strains belonging to B. animalis subsp. lactis and B. animalis subsp. animalis. Additional file 3: Figure S3: strains belonging to B. longum subsp. longum, B. longum subsp. infantis, B. longum subsp. suis). About 95% of the strains confirmed the taxonomic identification previously assigned. Two strains, B1955 and Su864, previously classified as B. catenulatum and B. longum subsp. suis respectively, gave different profiles from those expected. The RFLP profiles of B1955 turned out to be the same of B. adolescentis ATCC 15703 (T), the dichotomous key confirmed the assignment to the B. adolescentis species. In addition, Su864 was identified as a B. breve strain. These results were also verified through a species-specific PCR [14].

Conclusions

In this work a PCR-RFLP based method to identify Bifidobacterium spp. was developed and tested on strains belonging to different species. The technique could efficiently differentiate all the 25 species of Bifidobacterium genus and the subspecies belonging to B. pseudolongum and B. animalis, with the support of an easy-to-handle dichotomous key. The technique turned out to be fast and easy, and presented a potential value for a rapid preliminary identification of bifidobacterial isolates.

Abbreviations

PCR: Polymerase chain reaction; RFLP-PCR: Restriction fragment length polymorphism; HSP60: Heat-shock protein 60; rDNA: Ribosomal DNA; ARDRA: Amplified ribosomal DNA restriction analysis; DGGE: Denaturing gradient gel electrophoresis; ERIC-PCR: Enterobacterial repetitive intergenic consensus-PCR; RAPD: Random amplified polymorphic DNA; cpnDB: Chaperonin database; TPY medium: Tryptone phytone, yeast medium; BUSCoB: (Bologna University Scardovi Collection of Bifidobacteria).

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

LB conceived the study. LB, VS and ES carried out all the bioinformatics, RFLP analyses, DNA extractions and culture handling. VS conceived the dichotomous key. MM and PM provided some of the strains tested together with the extracted DNA. DDG and FG supervised the work. LB, VS, DDG and FG contributed to paper writing. All authors read and approved the final manuscript. BB supported the research.

Additional file 1: Figure S1

Example of agarose gel electrophoresis of hsp60 amplicons from different bifidobacterial strains.

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Additional file 2: Figure S2

Agarose gel electrophoresis of digested hsp60 DNA fragments with HaeIII (negative image). Lane1, ladder 20 bp (Sigma-Aldrich); Lane 2–6, B. animalis subsp.lactis strains Ra20, Ra18, F439, P23, P32; Lane 7–8, B. animalis subsp. animalis strains T169, T6/1; Lane 9, ladder 20 bp (Sigma-Aldrich).

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Additional file 3: Figure S3

Agarose gel electrophoresis of digested hsp60 DNA fragments with HaeIII (negative image). Lane1, ladder 20 bp (Sigma-Aldrich); Lane 2–4, B. longum subsp. suis strains Su864, Su908, Su932; Lane 5–6, B. longum subsp. longum strains PCB133, ATCC 15707 (T); Lane 7–9, B. longum subsp. infantis strains ATCC 15697 (T), B7740, B7710; Lane 9, ladder 20 bp (Sigma-Aldrich).

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