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Characterization of bifidobacteria suitable for probiotic use in calves
Anaerobe 18 (2012) 166e168
Contents lists available at SciVerse ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Note
Characterization of bifidobacteria suitable for probiotic use in calves ra Bunesová a, Konrad J. Domig b, Jirí Killer a, c, Eva Vlková a, *, Jan Kope Ve cný c, Jakub Mrázek c, a a ch Rada Sárka Ro cková , Vojte a
Department of Microbiology, Nutrition and Dietetics, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, Prague 6-Suchdol 16521, Czech Republic b BOKU - University of Natural Resources and Life Sciences, Vienna, Department of Food Science and Technology, Institute of Food Science, Muthgasse 18, A-1190 Vienna, Austria c ská 1083, Prague 4-Krc 14200, Czech Republic Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Víden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2011 Received in revised form 29 August 2011 Accepted 25 September 2011 Available online 1 October 2011
In our previous experiment, the ten calves originated bifidobacterial strains were administered to calves and re-isolated. Fingerprinting techniques used in this study enabled us to distinguish the surviving and non-surviving strains. Only the species Bifidobacterium animalis ssp. animalis and Bifidobacterium longum ssp. suis were found to survive in the intestine. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Bifidobacteria Identification Calves Probiotic 16S rRNA and hsp60 genes sequencing Fingerprint techniques
Feeding practices, composition of animal diets and farm management are parameters that can influence microbial balance in the gastrointestinal tract and consequently affect the feed efficiency, digestive welfare and health of the animals [1]. In young pre-ruminants, bacterial probiotics such as lactic acid bacteria, Propionibacterium spp., Bifidobacterium spp. or Bacillus spp. spores are used to improve animal health [2]. Probiotics generally target the intestine, because the rumen is not yet developed. Incorporation of probiotics in young calf diets prevents the occurrence of possible imbalances in the normal microbiota in the intestinal tract to improve the growth of young calves housed under stressful conditions by preventing diarrhoea [3]. In the intestinal tracts of animals and humans, bifidobacteria are considered as one of the key genera [4]. Several species are reported as a host specific [5]. Their presence in high numbers is associated with good health status of the host. The objective of this study was to identify and characterize bifidobacteria strains isolated from calf faeces by DNA fingerprinting techniques and sequencing. The second aim was to confirm the identity of re-isolated bifidobacteria after the probiotic treatment of calves. * Corresponding author. Tel.: þ420 2 24 38 27 55; fax: þ420 2 24 38 27 60. E-mail address: [email protected] (E. Vlková). 1075-9964/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2011.09.008
All bifidobacterial strains identified in this study were obtained from calf faeces during our previous experiment. The design of our previous experiment in brief was follows: The ten calf origin strains with suitable physiological properties were identified by sequencing. A mixture of rifampicin-resistant variant (RRBs) of these strains was fed to three 2-days-old suckling calves. The survival ability of strains administered was monitored by cultivation on selective agar with addition of rifampicin. The nine best survived strains (those which survived in intestinal tract of calves for at least 40 days in counts higher than 106 CFU g1) were re-isolated and identified to genus level via demonstration of fructose-6-phosphate phosphoketolase activity [6]. In this study, RAPD-PCR, REP-PCR, and the sequencing of the hsp60 gene were performed with all (original and re-isolated) strains. Additionally, the 16S rRNA gene was sequenced in re-isolated strains. DNA was isolated from the bacterial cells according to the standard procedure for Gram-positive bacteria (DNeasyÒ Blood & Tissue Kit, Qiagen, Germany). The type strains used as standards for fingerprinting techniques are Bifidobacterium animalis ssp. animalis LMG (Laboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium) 10508, Bifidobacterium thermophilum CUETM (Collection Unité Ecotoxicologie Microbienne, France) 89/ 40, Bifidobacterium choerinum CUETM 89/45 and Bifidobacterium
V. Bunesová et al. / Anaerobe 18 (2012) 166e168
longum ssp. suis CUETM 91/24. Randomly amplified polymorphic DNA (RAPD-PCR) using the primers OPA-2 [7], 103 and 173 [8], and extragenic palindromic DNA (REP-PCR) with primer (GTG)5 [9] were performed according the authors. RAPD-PCR and REP-PCR electrophoreticaly separated patterns were documented by digital imaging (Chemilmager 5500, Alpha Innotech, USA) and analyzed using Bionumerics software (Version 6.5, Applied Maths, Belgium) by Gevers et al. [9]. Sequencing of 16S rRNA and hsp60 genes were performed according to Killer et al. [10]. All sequences were deposited in GenBank nucleotide sequence databases using the Banklt program on the National Center for Biotechnology Information database (NCBI; www.ncbi.nlm.nih.gov) website. For comparison of the 16S rRNA genes sequences, the type strain of B. animalis ssp. animalis JCM (Japan Collection of Microorganisms, Wako, Japan) 1190T (D86185), B. longum ssp. suis ATCC (American Type Culture Collection, Rockville, MD, USA) 27533T (M58743), B. thermophilum ATCC 25525T (U10151) and B. choerinum ATCC 27686T (D86186) were used. In the case of partial hsp60 gene sequences, the standards used for comparison were sequences of B. animalis ssp. animalis JCM 1190T (AY004273) and B. longum ssp. suis JCM 1269T (AY013248). The strains obtained and identified by sequencing the 16S rRNA gene in our previous study [6] as B. animalis ssp. animalis (6 strains; 23II, 805III2, 805P4, 813P3, 12II1, 17III1), B. thermophilum (2 strains; 17III2, 25II), B. choerinum (1 strain; 23I2) and B. longum ssp. suis (1 strain; 22II) were identified to the same species additionally by sequencing of the hsp60 gene in the present study, with exception of B. choerinum 23I2 (Table 1). Sequencing results were confirmed by the REP-PCR and RAPD-PCR techniques, with the exception of two strains (12II1, 17III1) that showed similar profiles to B. thermophilum CUETM 89 (Fig. 1). To our knowledge, there are no recent studies describing the bifidobacterial composition in the intestine of calves on a milk diet, but it is generally stated that bifidobacteria are host specific [11]. The species B. thermophilum is common for calves on a milk diet [12]. B. animalis spp. animalis is widespread in various animal species [13]. Lukás et al. [14] detected B. animalis in faecal samples of calves by denaturing gradient gel electrophoresis from the first day of postnatal life. B. longum ssp. suis [15] and B. choerinum [16] are considered as host specific species for pigs. However, we found these species in faecal samples
167
of calves. Our results also show that some bifidobacterial species might not be host specific or have a broader host tolerance. One of the most important characteristics of probiotic bacteria is their ability to pass through the upper digestive tract of the host in sufficient quantity and to colonize the large intestine. The strains administered must clearly be distinguished from naturally occurring ones. Generally, fingerprinting techniques allow the differentiation of bifidobacteria to species or even strain level [17e19]. In our study, the highest similarity was seen (Fig. 1) between original (23II, 805III2, 805P4, and 813 P3) and surviving (1/10, 1/11, 3/9, 3/ 10, 4/9a, 4/10, and 5/10) strains of B. animalis spp. animalis (identified by sequencing, Table 1). These 11 stains showed identical profiles, were clustered in one group, and it can be stated that the B. animalis ssp. animalis strains administered were able survive in digestive tract of calves. Also, strains 4/9 and 5/9 that belonged by sequencing to subspecies B. longum ssp. suis showed the same profiles as the strain B. longum ssp. suis 22II from the bifidobacterial mixture used for probiotic treatment (Fig. 1). Another two strains of species B. animalis ssp. animalis (12II1 and 17III1), which were grouped separately from other B. animalis ssp. animalis strains, the strains B. thermophilum 17III2 and 25II and B. choerinum 23I2 were not able to survive in the digestive tract of treated calves, as these species/strains were not found among re-isolated strains (Table 1, Fig. 1). Our results showed that the colonization ability of probiotic bacteria could be species or even strain specific. The primers used in this study seem to be suitable for differentiation of bifidobacterial strains from the gastrointestinal tract of calves. As indicated in Fig. 1, the 23 tested strains were divided into four main clusters, which are in agreement with the species classification obtained by sequencing. The type strains of bifidobacteria used in the study were not of calf origin, therefore the similarities between the type and wild strains were lower. The strain B. animalis spp. animalis LMG 10508 was isolated from rat faeces, B. longum spp. suis CUETM 91/24, B. thermophilum CUETM 89/40 and B. choerinum CUETM 89/45 from pig faeces. The highest similarity between the type and wild strains was seen in B. longum spp. suis. Our new isolates may be differentiated from the type strain, other reference strains and commercial bifidobacteria strains by using RAPD-PCR and REP-PCR.
Table 1 Identification of calf bifidobacteria by fingerprint techniques and similarity of hsp60 and 16S rRNA (only in re-isolated strains, marked in bold) genes with the most closely related species of bifidobacteria. Strain code
Identification by hsp60 (16S rRNA) gene sequencing
Similarity of hsp60 (16S rRNA) genes with type strain of bifidobacterial species/subspecies (%)
Genbank accession nubmer of hsp60 (16S rRNA) gene
Identification by fingerprint techniques
23II 805III2 805P4 813P3 12II1 17III1 1/10 1/11 3/9 3/10 4/9a 4/10 5/10 17III2 25II 23I2 22II 4/9 5/9
B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. thermophilum B. thermophilum not identified B. longum ssp. suis B. longum ssp. suis B. longum ssp. suis
96.1 97.2 97.1 97.2 97.2 97.1 97.5 97.6 97.5 97.2 97.3 97.2 96.5 97.4 97.6
JF461067 JF461069 JF461070 JF461071 JF461064 JF461065 HQ851042 HQ851046 HQ851039 HQ851043 HQ851047 HQ851044 HQ851045 JF461066 JF461068
B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. thermophilum B. thermophilum B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. thermophilum B. thermophilum B. choerinum B. longum ssp. suis B. longum ssp. suis B. longum ssp. suis
(99.1) (99.4) (99.4) (99.4) (99.3) (99.4) (99.2)
98.3 98.3 (99.9) 98.1 (99.4)
(HQ851031) (HQ851035) (HQ851028) (HQ851032) (HQ851036) (HQ851033) (HQ851034)
JF461072 HQ851040 (HQ851029) HQ851041 (HQ851030)
V. Bunesová et al. / Anaerobe 18 (2012) 166e168
168
Fig. 1. Dendogram of tested Bifidobacterium strains based on REP-PCR and RAPD-PCR (for description of the strains see Table 1).
The fingerprint techniques used in this study were found to be suitable method for clustering bifidobacterial isolates and strainspecific identification, but not for the species determination of calf origin bifidobacteria. A more reliable method for bifidobacteria species identification is sequencing 16S rRNA and hsp60 genes. Using fingerprinting methods, the bifidobacterial strain, which were able to survive in the intestinal tract of calves, were clearly distinguished. These strains belonged to the species B. animalis ssp. animalis and B. longum ssp. suis. Acknowledgements The study was supported by grants GACR 523/08/1091, GACR 525/08/H060, and MSM 60460709/01. References [1] Chaucheyras-Durand F, Durand H. Probiotics in animal nutrition and health. Beneficial Microbe 2010;1:3e9. [2] Chaucheyras-Durand F, Fonty G. Influence of a probiotic yeast (Saccharomyces cerevisiae CNCM I-1077) on microbial colonization and fermentation in the rumen of newborn lambs. Microb Ecol Health Dis 2002;14:30e6. [3] Frizzo LS, Soto LP, Zbrun MV, Bertozzi E, Sequeira G, Rodríguez Armesto R, et al. Lactic acid bacteria to improve growth performance in young calves fed milk replacer and spray-dried whey powder. Anim Feed Sci Tech 2010;157: 159e67. [4] Rada V, Vlková E, Nevoral J, Trojanová I. Comparison of bacterial flora and enzymatic activity in faeces of infants and calves. FEMS Microbiol Lett 2006; 258:25e8. [5] Biavati B, Mattarelli P. The family bifidobacteriaceae. In: Dworkin M, Hansen PA, Lessel EF, editors. The prokaryotes: archaea, bacteria: firmicutes, actinomycetes, vol. 3. New York: Springer-Verlag; 2006. p. 322e82. [6] Vlková E, Grmanová M, Mrázek J, Killer J, Kope cný J, Bunesová V, et al. Survival of bifidobacteria administered to calves. Folia Microbiol 2010;55:390e2.
[7] Mättö J, Malinen E, Suihko ML, Alander M, Palva A, Saarela M. Genetic heterogeneity and functional properties of intestinal bifidobacteria. J Appl Microbiol 2004;97:459e70. [8] Sakata S, Kitahara M, Sakamoto M, Hayashi H, Fukuyama M, Benno Y. Unification of Bifidobacterium infantis and Bifidobacterium suis as Bifidobacterium longum. Int J Syst Evol Microbiol 2002;52:1945e51. [9] Gevers D, Huys G, Swings J. Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiol Lett 2001;205:31e6. [10] Killer J, Kope cný J, Mrázek J, Havlík J, Koppová I, Benada O, et al. Bombiscardovia coagulans gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of bumblebees. Syst Appl Microbiol 2010;33:359e66. [11] Fuller R. Probiotics 2: applications and practical aspects. London: Chapman and Hall; 1997. pp. 1e9. [12] Mitsuoka T. Vergleichende Untersuchungen über die Bifidobakterien aus dem Verdauungstarakt von Menchen und Tieren. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Abt Orig 1969;A210:52e64. [13] Scardovi V. Genus Bifidobacterium Orla-Jensen. 1924, 472. In: Krieg NR, Holt JG, editors. Bergey’s manual of systematic bacteriology, vol. 2. Baltimore, MD, USA: Williams and Wilkins; 1986. p. 1418e34. [14] Lukás F, Koppová I, Kudrna V, Kope cný J. Postnatal development of bacterial population in the gastrointestinal tract of calves. Folia Microbiol 2007;52: 99e104. [15] Matteuzzi D, Crociani F, Zani G, Trovatelli LD. Bifidobacterium suis n. sp.: a new species of the genus bifidobacterium isolated from pig feces. Z Allg Mikrobiol 1971;11:387e95. [16] Scardovi V, Casalicchio F, Vincenzi N. Multiple electrophoretic forms of transaldolase and 6-phosphogluconic dehydro-genase and their relationships to the taxonomy and ecology of the bifidobacteria. Int J Syst Bacteriol 1979; 29:312e27. [17] Mayer HK, Amtmann E, Phlilippi E, Steinegger G, Mayrhofer S, Kneifel W. Molecular discrimination of new isolates of Bifidobacterium animalis subsp. lactis from reference strains and commercial probiotic strains. Int Dairy J 2007;17:565e73. [18] Vincent D, Roy D, Mondou F, Déry C. Characterization of bifidobacteria by random DNA amplification. Int J Food Microbiol 1998;43:185e93. [19] Du Plessis EM, Dicks LMT. Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. Curr Microbiol 1995;31:114e8.
Contents lists available at SciVerse ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Note
Characterization of bifidobacteria suitable for probiotic use in calves ra Bunesová a, Konrad J. Domig b, Jirí Killer a, c, Eva Vlková a, *, Jan Kope Ve cný c, Jakub Mrázek c, a a ch Rada Sárka Ro cková , Vojte a
Department of Microbiology, Nutrition and Dietetics, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, Prague 6-Suchdol 16521, Czech Republic b BOKU - University of Natural Resources and Life Sciences, Vienna, Department of Food Science and Technology, Institute of Food Science, Muthgasse 18, A-1190 Vienna, Austria c ská 1083, Prague 4-Krc 14200, Czech Republic Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Víden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2011 Received in revised form 29 August 2011 Accepted 25 September 2011 Available online 1 October 2011
In our previous experiment, the ten calves originated bifidobacterial strains were administered to calves and re-isolated. Fingerprinting techniques used in this study enabled us to distinguish the surviving and non-surviving strains. Only the species Bifidobacterium animalis ssp. animalis and Bifidobacterium longum ssp. suis were found to survive in the intestine. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Bifidobacteria Identification Calves Probiotic 16S rRNA and hsp60 genes sequencing Fingerprint techniques
Feeding practices, composition of animal diets and farm management are parameters that can influence microbial balance in the gastrointestinal tract and consequently affect the feed efficiency, digestive welfare and health of the animals [1]. In young pre-ruminants, bacterial probiotics such as lactic acid bacteria, Propionibacterium spp., Bifidobacterium spp. or Bacillus spp. spores are used to improve animal health [2]. Probiotics generally target the intestine, because the rumen is not yet developed. Incorporation of probiotics in young calf diets prevents the occurrence of possible imbalances in the normal microbiota in the intestinal tract to improve the growth of young calves housed under stressful conditions by preventing diarrhoea [3]. In the intestinal tracts of animals and humans, bifidobacteria are considered as one of the key genera [4]. Several species are reported as a host specific [5]. Their presence in high numbers is associated with good health status of the host. The objective of this study was to identify and characterize bifidobacteria strains isolated from calf faeces by DNA fingerprinting techniques and sequencing. The second aim was to confirm the identity of re-isolated bifidobacteria after the probiotic treatment of calves. * Corresponding author. Tel.: þ420 2 24 38 27 55; fax: þ420 2 24 38 27 60. E-mail address: [email protected] (E. Vlková). 1075-9964/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2011.09.008
All bifidobacterial strains identified in this study were obtained from calf faeces during our previous experiment. The design of our previous experiment in brief was follows: The ten calf origin strains with suitable physiological properties were identified by sequencing. A mixture of rifampicin-resistant variant (RRBs) of these strains was fed to three 2-days-old suckling calves. The survival ability of strains administered was monitored by cultivation on selective agar with addition of rifampicin. The nine best survived strains (those which survived in intestinal tract of calves for at least 40 days in counts higher than 106 CFU g1) were re-isolated and identified to genus level via demonstration of fructose-6-phosphate phosphoketolase activity [6]. In this study, RAPD-PCR, REP-PCR, and the sequencing of the hsp60 gene were performed with all (original and re-isolated) strains. Additionally, the 16S rRNA gene was sequenced in re-isolated strains. DNA was isolated from the bacterial cells according to the standard procedure for Gram-positive bacteria (DNeasyÒ Blood & Tissue Kit, Qiagen, Germany). The type strains used as standards for fingerprinting techniques are Bifidobacterium animalis ssp. animalis LMG (Laboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium) 10508, Bifidobacterium thermophilum CUETM (Collection Unité Ecotoxicologie Microbienne, France) 89/ 40, Bifidobacterium choerinum CUETM 89/45 and Bifidobacterium
V. Bunesová et al. / Anaerobe 18 (2012) 166e168
longum ssp. suis CUETM 91/24. Randomly amplified polymorphic DNA (RAPD-PCR) using the primers OPA-2 [7], 103 and 173 [8], and extragenic palindromic DNA (REP-PCR) with primer (GTG)5 [9] were performed according the authors. RAPD-PCR and REP-PCR electrophoreticaly separated patterns were documented by digital imaging (Chemilmager 5500, Alpha Innotech, USA) and analyzed using Bionumerics software (Version 6.5, Applied Maths, Belgium) by Gevers et al. [9]. Sequencing of 16S rRNA and hsp60 genes were performed according to Killer et al. [10]. All sequences were deposited in GenBank nucleotide sequence databases using the Banklt program on the National Center for Biotechnology Information database (NCBI; www.ncbi.nlm.nih.gov) website. For comparison of the 16S rRNA genes sequences, the type strain of B. animalis ssp. animalis JCM (Japan Collection of Microorganisms, Wako, Japan) 1190T (D86185), B. longum ssp. suis ATCC (American Type Culture Collection, Rockville, MD, USA) 27533T (M58743), B. thermophilum ATCC 25525T (U10151) and B. choerinum ATCC 27686T (D86186) were used. In the case of partial hsp60 gene sequences, the standards used for comparison were sequences of B. animalis ssp. animalis JCM 1190T (AY004273) and B. longum ssp. suis JCM 1269T (AY013248). The strains obtained and identified by sequencing the 16S rRNA gene in our previous study [6] as B. animalis ssp. animalis (6 strains; 23II, 805III2, 805P4, 813P3, 12II1, 17III1), B. thermophilum (2 strains; 17III2, 25II), B. choerinum (1 strain; 23I2) and B. longum ssp. suis (1 strain; 22II) were identified to the same species additionally by sequencing of the hsp60 gene in the present study, with exception of B. choerinum 23I2 (Table 1). Sequencing results were confirmed by the REP-PCR and RAPD-PCR techniques, with the exception of two strains (12II1, 17III1) that showed similar profiles to B. thermophilum CUETM 89 (Fig. 1). To our knowledge, there are no recent studies describing the bifidobacterial composition in the intestine of calves on a milk diet, but it is generally stated that bifidobacteria are host specific [11]. The species B. thermophilum is common for calves on a milk diet [12]. B. animalis spp. animalis is widespread in various animal species [13]. Lukás et al. [14] detected B. animalis in faecal samples of calves by denaturing gradient gel electrophoresis from the first day of postnatal life. B. longum ssp. suis [15] and B. choerinum [16] are considered as host specific species for pigs. However, we found these species in faecal samples
167
of calves. Our results also show that some bifidobacterial species might not be host specific or have a broader host tolerance. One of the most important characteristics of probiotic bacteria is their ability to pass through the upper digestive tract of the host in sufficient quantity and to colonize the large intestine. The strains administered must clearly be distinguished from naturally occurring ones. Generally, fingerprinting techniques allow the differentiation of bifidobacteria to species or even strain level [17e19]. In our study, the highest similarity was seen (Fig. 1) between original (23II, 805III2, 805P4, and 813 P3) and surviving (1/10, 1/11, 3/9, 3/ 10, 4/9a, 4/10, and 5/10) strains of B. animalis spp. animalis (identified by sequencing, Table 1). These 11 stains showed identical profiles, were clustered in one group, and it can be stated that the B. animalis ssp. animalis strains administered were able survive in digestive tract of calves. Also, strains 4/9 and 5/9 that belonged by sequencing to subspecies B. longum ssp. suis showed the same profiles as the strain B. longum ssp. suis 22II from the bifidobacterial mixture used for probiotic treatment (Fig. 1). Another two strains of species B. animalis ssp. animalis (12II1 and 17III1), which were grouped separately from other B. animalis ssp. animalis strains, the strains B. thermophilum 17III2 and 25II and B. choerinum 23I2 were not able to survive in the digestive tract of treated calves, as these species/strains were not found among re-isolated strains (Table 1, Fig. 1). Our results showed that the colonization ability of probiotic bacteria could be species or even strain specific. The primers used in this study seem to be suitable for differentiation of bifidobacterial strains from the gastrointestinal tract of calves. As indicated in Fig. 1, the 23 tested strains were divided into four main clusters, which are in agreement with the species classification obtained by sequencing. The type strains of bifidobacteria used in the study were not of calf origin, therefore the similarities between the type and wild strains were lower. The strain B. animalis spp. animalis LMG 10508 was isolated from rat faeces, B. longum spp. suis CUETM 91/24, B. thermophilum CUETM 89/40 and B. choerinum CUETM 89/45 from pig faeces. The highest similarity between the type and wild strains was seen in B. longum spp. suis. Our new isolates may be differentiated from the type strain, other reference strains and commercial bifidobacteria strains by using RAPD-PCR and REP-PCR.
Table 1 Identification of calf bifidobacteria by fingerprint techniques and similarity of hsp60 and 16S rRNA (only in re-isolated strains, marked in bold) genes with the most closely related species of bifidobacteria. Strain code
Identification by hsp60 (16S rRNA) gene sequencing
Similarity of hsp60 (16S rRNA) genes with type strain of bifidobacterial species/subspecies (%)
Genbank accession nubmer of hsp60 (16S rRNA) gene
Identification by fingerprint techniques
23II 805III2 805P4 813P3 12II1 17III1 1/10 1/11 3/9 3/10 4/9a 4/10 5/10 17III2 25II 23I2 22II 4/9 5/9
B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. thermophilum B. thermophilum not identified B. longum ssp. suis B. longum ssp. suis B. longum ssp. suis
96.1 97.2 97.1 97.2 97.2 97.1 97.5 97.6 97.5 97.2 97.3 97.2 96.5 97.4 97.6
JF461067 JF461069 JF461070 JF461071 JF461064 JF461065 HQ851042 HQ851046 HQ851039 HQ851043 HQ851047 HQ851044 HQ851045 JF461066 JF461068
B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. thermophilum B. thermophilum B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. thermophilum B. thermophilum B. choerinum B. longum ssp. suis B. longum ssp. suis B. longum ssp. suis
(99.1) (99.4) (99.4) (99.4) (99.3) (99.4) (99.2)
98.3 98.3 (99.9) 98.1 (99.4)
(HQ851031) (HQ851035) (HQ851028) (HQ851032) (HQ851036) (HQ851033) (HQ851034)
JF461072 HQ851040 (HQ851029) HQ851041 (HQ851030)
V. Bunesová et al. / Anaerobe 18 (2012) 166e168
168
Fig. 1. Dendogram of tested Bifidobacterium strains based on REP-PCR and RAPD-PCR (for description of the strains see Table 1).
The fingerprint techniques used in this study were found to be suitable method for clustering bifidobacterial isolates and strainspecific identification, but not for the species determination of calf origin bifidobacteria. A more reliable method for bifidobacteria species identification is sequencing 16S rRNA and hsp60 genes. Using fingerprinting methods, the bifidobacterial strain, which were able to survive in the intestinal tract of calves, were clearly distinguished. These strains belonged to the species B. animalis ssp. animalis and B. longum ssp. suis. Acknowledgements The study was supported by grants GACR 523/08/1091, GACR 525/08/H060, and MSM 60460709/01. References [1] Chaucheyras-Durand F, Durand H. Probiotics in animal nutrition and health. Beneficial Microbe 2010;1:3e9. [2] Chaucheyras-Durand F, Fonty G. Influence of a probiotic yeast (Saccharomyces cerevisiae CNCM I-1077) on microbial colonization and fermentation in the rumen of newborn lambs. Microb Ecol Health Dis 2002;14:30e6. [3] Frizzo LS, Soto LP, Zbrun MV, Bertozzi E, Sequeira G, Rodríguez Armesto R, et al. Lactic acid bacteria to improve growth performance in young calves fed milk replacer and spray-dried whey powder. Anim Feed Sci Tech 2010;157: 159e67. [4] Rada V, Vlková E, Nevoral J, Trojanová I. Comparison of bacterial flora and enzymatic activity in faeces of infants and calves. FEMS Microbiol Lett 2006; 258:25e8. [5] Biavati B, Mattarelli P. The family bifidobacteriaceae. In: Dworkin M, Hansen PA, Lessel EF, editors. The prokaryotes: archaea, bacteria: firmicutes, actinomycetes, vol. 3. New York: Springer-Verlag; 2006. p. 322e82. [6] Vlková E, Grmanová M, Mrázek J, Killer J, Kope cný J, Bunesová V, et al. Survival of bifidobacteria administered to calves. Folia Microbiol 2010;55:390e2.
[7] Mättö J, Malinen E, Suihko ML, Alander M, Palva A, Saarela M. Genetic heterogeneity and functional properties of intestinal bifidobacteria. J Appl Microbiol 2004;97:459e70. [8] Sakata S, Kitahara M, Sakamoto M, Hayashi H, Fukuyama M, Benno Y. Unification of Bifidobacterium infantis and Bifidobacterium suis as Bifidobacterium longum. Int J Syst Evol Microbiol 2002;52:1945e51. [9] Gevers D, Huys G, Swings J. Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiol Lett 2001;205:31e6. [10] Killer J, Kope cný J, Mrázek J, Havlík J, Koppová I, Benada O, et al. Bombiscardovia coagulans gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of bumblebees. Syst Appl Microbiol 2010;33:359e66. [11] Fuller R. Probiotics 2: applications and practical aspects. London: Chapman and Hall; 1997. pp. 1e9. [12] Mitsuoka T. Vergleichende Untersuchungen über die Bifidobakterien aus dem Verdauungstarakt von Menchen und Tieren. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Abt Orig 1969;A210:52e64. [13] Scardovi V. Genus Bifidobacterium Orla-Jensen. 1924, 472. In: Krieg NR, Holt JG, editors. Bergey’s manual of systematic bacteriology, vol. 2. Baltimore, MD, USA: Williams and Wilkins; 1986. p. 1418e34. [14] Lukás F, Koppová I, Kudrna V, Kope cný J. Postnatal development of bacterial population in the gastrointestinal tract of calves. Folia Microbiol 2007;52: 99e104. [15] Matteuzzi D, Crociani F, Zani G, Trovatelli LD. Bifidobacterium suis n. sp.: a new species of the genus bifidobacterium isolated from pig feces. Z Allg Mikrobiol 1971;11:387e95. [16] Scardovi V, Casalicchio F, Vincenzi N. Multiple electrophoretic forms of transaldolase and 6-phosphogluconic dehydro-genase and their relationships to the taxonomy and ecology of the bifidobacteria. Int J Syst Bacteriol 1979; 29:312e27. [17] Mayer HK, Amtmann E, Phlilippi E, Steinegger G, Mayrhofer S, Kneifel W. Molecular discrimination of new isolates of Bifidobacterium animalis subsp. lactis from reference strains and commercial probiotic strains. Int Dairy J 2007;17:565e73. [18] Vincent D, Roy D, Mondou F, Déry C. Characterization of bifidobacteria by random DNA amplification. Int J Food Microbiol 1998;43:185e93. [19] Du Plessis EM, Dicks LMT. Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. Curr Microbiol 1995;31:114e8.