PCR; hsp60 gene; Bifidobacterium; detection; fecal indicators; raw milk; raw milk cheese; mupirocin
Abstract :
[en] Bifidobacteria are well known for their beneficial effects on health and are used as probiotics in food and pharmaceutical products. As they form one of the most important groups in both human and animal feces, their use as fecal indicator organisms in raw milk products has recently been proposed. Bifidobacteria species isolated in humans are different from those isolated in animals. It should therefore be possible to determine contamination origin (human or animal). A method of detecting the Bifidobacterium genus was developed by PCR targeting the hsp60 gene. The genus Bifidobacterium was identified by PCR amplification of a 217-bp hsp60 gene fragment. The degenerated primer pair specific to the Bifidobacterium genus used was tested for it specificity on 127 strains. Sensitivity was measured on artificially contaminated samples. Food can however be a difficult matrix for PCR testing since it contains PCR inhibitors. So an internal PCR control was used. An artificially created DNA fragment of 315 bp was constructed. The PCR detection method was tested on raw milk and cheese samples and compared with three culture-based methods, which comprised enrichment and isolation steps. The enrichment step used Brain Heart Infusion medium with propionic acid, iron citrate, yeast extract, supplemented with mupirocin (BHMup) or not (BH) and the isolation step used Columbia blood agar medium, supplemented with mupirocin (CMup) or not (C). The method using mupirocin at both enrichment and isolation steps and the PCR method performed from the culture in BHMup enrichment medium were shown to be the most efficient. No significant difference was observed in raw milk samples between PCR from BHMup and the culture-based method BHMup/CMup, while a significant difference was noticed between the same methods in raw milk cheese samples, which would favor using PCR. The results suggested that PCR on the hsp60 gene was convenient for a rapid detection of bifidobacteria in raw milk and raw milk cheese samples and that bifidobacteria always present throughout raw milk cheese production could be efficiently used as fecal indicators.
Disciplines :
Microbiology Food science
Author, co-author :
Delcenserie, Véronique ; Université de Liège - ULiège > Département de sciences des denrées alimentaires > Gestion de la qualité dans la chaîne alimentaire
Bechoux, Nathalie ; Université de Liège - ULiège > Département de sciences des denrées alimentaires > Microbiologie des denrées alimentaires
China, Bernard
Daube, Georges ; Université de Liège - ULiège > Département de sciences des denrées alimentaires > Microbiologie des denrées alimentaires
Gavini, Françoise; Institut Scientifique de Recherche Agronomique - INRA
Language :
English
Title :
A PCR method for detection of bifidobacteria in raw milk and raw milk cheese: comparison with culture-based methods
Publication date :
2005
Journal title :
Journal of Microbiological Methods
ISSN :
0167-7012
eISSN :
1872-8359
Publisher :
Elsevier Science Bv, Amsterdam, Netherlands
Volume :
61
Issue :
1
Pages :
55-67
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
DG RDT - Commission Européenne. Direction Générale de la Recherche et de l'Innovation [BE]
H. Beerens An elective and selective isolation medium for Bifidobacterium spp. Lett. Appl. Microbiol. 11 1990 155 157
H. Beerens Bifidobacteria as indicators of faecal contamination in meat and meat products. Detection, determination of origin and comparison with Escherichia coli Int. J. Food Microbiol. 40 1998 203 207
H. Beerens, B. Hass Brac de la Perriere, and F. Gavini Evaluation of the hygienic quality of raw milk based on the presence of bifidobacteria: the cow as a source of faecal contamination Int. J. Food Microbiol. 54 2000 163 169
A. Bernhard, and K. Field Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes Appl. Environ. Microbiol. 66 2000 1587 1594
B. Biavati, M. Vescovo, S. Torriani, and V. Bottazi Bifidobacteria: history, ecology, physiology and applications Ann. Microbiol. 50 2000 117 131
X. Bonjoch, E. Balleste, and A. Blanch Multiplex PCR with 16S rRNA gene-targeted primers of Bifidobacterium spp. to identify sources of fecal pollution Appl. Environ. Microbiol. 70 2004 3171 3175
P. Brigidi, E. Swennen, B. Vitali, M. Rossi, and D. Matteuzzi PCR detection of Bifidobacterium strains and Streptococcus thermophilus in feces of human subjects after oral bacteriotherapy and yogurt consumption Int. J. Food Microbiol. 81 2003 203 209
P. Dagnelie Analyse de la variance à un critère Les Presses Agronomiques de Gembloux Théorie et Méthodes Statistiques vol. 2 1975 126 138
V. Delcenserie, N. Bechoux, T. Leonard, B. China, and G. Daube Discrimination between Bifidobacterium species from human and animal origin by PCR-Restriction fragment length polymorphism J. Food Prot. 67 2004 1284 1288
S. Fasoli, M. Marzotto, L. Rizzotti, F. Rossi, F. Dellaglio, and S. Torriani Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis Int. J. Food Microbiol. 82 2003 59 70
F. Gavini, and H. Beerens Origin and identification of bifidobacteria strains isolated from meat and meat products Int. J. Food Microbiol. 46 1999 81 85
F. Gavini, AM. Pourcher, C. Neut, D. Monget, C. Romond, C. Oger, and D. Izard Phenotypic differentiation of bifidobacteria of human and animal origins Int. J. Syst. Bacteriol. 41 1991 548 557
B. Gilpin, T. James, F. Nourozi, D. Saunders, P. Scholes, and M. Savill The use of chemical and molecular microbial indicators for faecal source identification Water Sci. Technol. 47 2003 39 43
M. Grand, M. Küffer, and A. Baumgartner Quantitative analysis and molecular identification of bifidobacteria strains in probiotic milk products Eur. Food Res. Technol. 217 2003 90 92
W. Jian, L. Zhu, and X. Dong New approach to phylogenetic analysis of the genus Bifidobacterium based on partial hsp60 gene sequences Int. J. Syst. Evol. Microbiol. 51 2001 1633 1638
W. Jian, and X. Dong Amplification of bacterial heat shock protein 60 gene using inverse PCR method Wei Sheng Wu Xue Bao 42 2002 56 61 (Chinese)
P. Leroy, and F. Farnir Le Chi-Carré dans les tests de conformité Les Editions de l'Université de Liège Méthodes Statistiques en Médecine Vétérinaire vol. 1 2000 120 124
P. Lynch, B. Gilpin, L. Sinton, and M. Savill The detection of Bifidobacterium adolescentis by colony hybridization as an indicator of human faecal pollution J. Appl. Microbiol. 92 2002 526 533
E. Malinen, J. Matto, M. Salmitie, M. Alander, M. Saarela, and A. Palva PCR-ELISA II: Analysis of Bifidobacterium populations in human faecal samples from a consumption trial with Bifidobacterium lactis Bb-12 and a galacto-oligosaccharide preparation Syst. Appl. Microbiol. 25 2002 249 258 (2003. Erratum in: Syst. Appl. Microbiol. 26, 154-155)
B. Martineau Comparison of four media for the selection of bifidobacteria in dog fecal samples Anaerobe 5 1999 123 127
T. Matsuki, K. Watanabe, J. Fujimoto, Y. Miyamoto, T. Takada, K. Matsumoto, H. Oyaizu, and R. Tanaka Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces Appl. Environ. Microbiol. 68 2002 5445 5451
T. Matsuki, K. Watanabe, R Tanaka, and H. Oyaizu Rapid identification of human intestinal bifidobacteria by 16S rRNA-targeted species- and group-specific primers FEMS Microbiol. Lett. 167 1998 113 121
T. Matsuki, K. Watanabe, R. Tanaka, M. Fukuda, and H. Oyaizu Distribution of bifidobacterial species in human intestinal microflora examined with 16S rRNA-gene-targeted species-specific primers Appl. Environ. Microbiol. 65 1999 4506 4512
L. Mikkelsen, C. Bendixen, M. Jakobsen, and B. Jensen Enumeration of bifidobacteria in gastrointestinal samples from piglets Appl. Environ. Microbiol. 69 2003 654 658
C. Mullié, M. Odou, E. Singer, M. Romond, and D. Izard Multiplex PCR using 16S rRNA gene-targeted primers for the identification of bifidobacteria from human origin FEMS Microbiol. Lett. 222 2003 129 136
Y. Nebra, and R. Blanch A new selective Medium for Bifidobacterium spp. Appl. Environ. Microbiol. 65 1999 5173 5176
Y. Nebra, X. Bonjoch, and A. Blanch Use of Bifidobacterium dentium as an indicator of the origin of fecal water pollution Appl. Environ. Microbiol. 69 2003 2651 2656
B. Pacher, and W. Kneifel Development of a culture medium for the detection and enumeration of bifidobacteria in fermented milk products Int. Dairy J. 6 1996 43 64
J. Payne, A. Morris, and P. Beers Note: evaluation of selective media for the enumeration of Bifidobacterium sp. in milk J. Appl. Microbiol. 86 1999 353 358
J. Petr, and V. Rada Bifidobacteria are obligate inhabitants of the crop of adult laying hens J. Vet. Med., B. Infect. Dis. Vet. Public Health 48 2001 227 233
V. Rada, and J. Petr A new selective medium for the isolation of glucose non-fermenting bifidobacteria from hen caeca J. Microbiol. Methods 43 2000 127 132
V. Rada, K. Sirotek, and J. Petr Evaluation of selective media for bifidobacteria in poultry and rabbit caecal samples Zentralbl. Veterinärmed. Beih. 46 1999 369 373
T. Requena, J. Burton, T. Matsuki, K. Munro, M.A. Simon, R. Tanaka, K. Watanabe, and G. Tannock Identification, detection and enumeration of human Bifidobacterium species by PCR targeting the transaldolase gene Appl. Environ. Microbiol. 68 2002 2420 2427
M. Rhodes, and H. Kator Sorbitol-fermenting bifidobacteria as indicators of diffuse human faecal pollution in estuarine watersheds J. Appl. Microbiol. 87 1999 528 535
D. Roy, P. Ward, and G. Champagne Differentiation of bifidobacteria by use of pulsed-field gel electrophoresis and polymerase chain reaction Int. J. Food Microbiol. 29 1996 11 29
D. Roy, and S. Sirois Molecular differentiation of Bifidobacterium species with amplified ribosomal DNA restriction analysis and alignment of short regions of the Idh gene FEMS Microbiol. Lett. 191 2000 17 24
V. Scardovi Genus Bifidobacterium P. Sneath N. Mair M. Sharpe J. Holt Bergey's manual of systematic bacteriology 1986 Williams and Wilkins Baltimore 1418 1434
P. Simpson, R. Ross, G. Fitzgerald, and C. Stanton Bifidobacterium psychraerophilum sp. nov. and Aeriscardovia aeriphila gen. nov., sp., nov., isolated from a porcine caecum Int. J. Syst. Evol. Microbiol. 54 2004 401 406
P. Simpson, G. Fitzgerald, C. Stanton, and R. Ross The evaluation of a mupirocin-based selective medium for the enumeration of bifidobacteria from probiotic animal feed J. Microbiol. Methods 57 2004 9 16
R.A. Sueiro, M. Araujo, C.J. Santos, M.J. Gomes, and M.J. Garrido Evaluation of Coli-ID and MUG Plus media for recovering Escherichia coli and other coliform bacteria from groundwater samples Water Sci. Technol. 43 2001 213 216
R. Sutherland, R. Boon, K. Griffin, P. Masters, B. Slocombe, and A. White Antibacterial activity of mupirocin (pseudomonic acid), a new antibiotic for topical use Antimicrob. Agents Chemother. 27 1985 495 498
K. Venema, and A. Maathuis A PCR-based method for identification of bifidobacteria from the human alimentary tract at the species level FEMS Microbiol. Lett. 224 2003 143 149