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Comparison of phenotypic and genotypic taxonomic methods for the identification of dairy enterococci

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Abstract

The purpose of the study was to assess the phenotypic and genotypic taxonomic congruence in order to allow species allocation of dairy enterococci. A total of 364 enterococci isolated from ewes'milk and cheese from four Portuguese Registered Designation of Origin areas and 25 type and reference strains of Enterococcus spp. were characterized by a polyphasic taxonomical approach involving 40 physiological and biochemical tests, whole-cell protein profiles, amplification of 16S-23S intergenic spacer regions (ITS-PCR) and subsequent restriction analysis (ARDRA). Ribotyping was also performed with reference strains and a subset of 146 isolates. Numerical hierarchic data analysis showed that single-technique identification levels increase from the physiological and biochemical tests to the protein approach, being lower with ITS/ARDRA and ribotyping. Cross-analysis confirmed a higher unmatching level in all pairwise combinations involving physiological and biochemical data. Whole-cell protein profiles followed by ITS/ARDRA identified 89% of the enterococci. Reliable identification of enterococci from milk and cheese could be obtained by analysis of whole-cell protein profiles. ITS-PCR can be used to confirm E. durans and E. faecium and ARDRA further confirms E. faecalis. Results revealed E. faecalis, E. durans, E. hirae and E. faecium as the prevalent species, although species prevalence showed some degree of variation among the areas.

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References

  • Baele M., Baele P., Vaneechoutte M., Storms V., Butaye P., Devriese L.A., Verschraegen G., Gillis M. and Haesebrouck F. 2000. Application of tRNA intergenic spacer PCR for identification of Enterococcus species. J. Clin. Microbiol. 38: 4201-4207.

    Google Scholar 

  • Colwell R.R. 1970. Polyphasic taxonomy of the genus Vibrio: numerical taxonomy of Vibrio cholerae, Vibrio parahaemolyticus and related Vibrio species. J. Bacteriol. 104: 410-433.

    Google Scholar 

  • de Angelis M., Corsetti A., Tosti N., Rossi J., Corbo M.R. and Gobbetti M. 2001. Characterization of non-starter lactic acid bacteria from Italian ewe cheeses based on phenotypic, genotypic, and cell wall protein analysis. Appl. Environ. Microb. 67: 2021-2020.

    Google Scholar 

  • Descheemaeker P., Lammens C., Pot B., Vandamme P. and Goossens H. 1997. Evaluation of arbitrarily primed PCR analysis and pulsed-field gel electrophoresis of large Genomic DNA fragments for identification of Enterococci important in human medicine. Int. J. Syst. Bacteriol. 47: 555-561.

    Google Scholar 

  • Devriese L.A., Pot B. and Collins M.D. 1993. Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups. J. Appl. Bacteriol. 75: 399-408.

    Google Scholar 

  • Devriese L.A. and Pot B. 1995. The genus Enterococcus. In: Wood B.J. and Holzapfel W.H. (eds), The Genera of Lactic Acid Bacteria. Blackie Academic and Professional, London, UK, pp. 327-367.

    Google Scholar 

  • Devriese L.A., Pot B., Van Damme L., Kersters K. and Haesebrouck F. 1995. Identification of Enterococcus species isolated from foods of animal origin. Int. J. Food Microbiol. 26: 187-197.

    Google Scholar 

  • DuPraw E.J. 1964. Non-linnean taxonomy. Nature 202: 849-852.

    Google Scholar 

  • Facklam R.R. and Collins M.D. 1989. Identification of Enterococcus species isolated from human infections by a conventional test scheme. J. Clin. Microbiol. 27: 731-734.

    Google Scholar 

  • Franz C.M., Holzapfel W.H. and Stiles M.E. 1999. Enterococci at the crossroads of food safety? Int. J. Food Microbiol. 47: 1-24.

    Google Scholar 

  • Freitas A.C., Pais C., Malcata F.X. and Hogg T.A. 1995. Microbiological characterization of Picante de Beira Baixa cheese. J. Food Protect. 59: 155-160.

    Google Scholar 

  • Gillis M., Vandamme P., De Vos P., Swings J. and Kersters K. 2001. Polyphasic taxonomy. In: Boone D.R. and Castenholz R.W. (eds), Garrity G.M. (Ed-in-chief) Bergey's Manual of Systematic Bacteriology, Vol 1. Springer-Verlag, New York, USA, pp. 43-48.

    Google Scholar 

  • Giraffa G. 2002. Enterococci in foods. FEMS Microbiol. Rev. 26: 163-171.

    Google Scholar 

  • Goh S.H., Facklam R.R., Chang M., Hill J.E., Tyrrell G.J., Burns E.C.M., Chan D., He C., Rathim T., Shaw C. and Hemmingsen S.M. 2000. Identification of Enterococcus species and phenotypically similar Lactococcus and Vagococcus species by reverse checkerboard hybridization to chaperonin 60 gene sequences. J. Clin. Microbiol. 38: 3953-3959.

    Google Scholar 

  • Grimont F. and Grimont P.A.D. 1986. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann. Inst. Pasteur/Microbiol. 137: 165-175.

    Google Scholar 

  • Jett B., Huycke M.M. and Gilmore M.S. 1994. Virulence of enterococci. Clin. Microbiol. Rev. 7: 162-478.

    Google Scholar 

  • Johnson A.P. 1994. The pathogenicity of enterococci. J. Antimicrob. Chemoth. 33: 1083-1089.

    Google Scholar 

  • Ke D., Picard F.J., Martineau F., Ménard C., Roy P.H., Ouellette M. and Bergeron M.G. 1999. Development of a PCR assay for rapid detection of enterococci. J. Clin. Microbiol. 37: 3497-3503.

    Google Scholar 

  • Knudtson L.M. and Hartman P.A. 1992. Routine procedures for isolation and identification of enterococci and fecal streptococci. Appl. Environ. Microb. 58: 3027-3031.

    Google Scholar 

  • Kostman J.R., Alden M.B., Mair M., Edlind T.D., LiPuma J.J. and Stull T.L. 1995. A universal approach to bacterial molecular epidemiology by polymerase chain reaction ribotyping. J. Infect. Dis. 171: 204-208.

    Google Scholar 

  • Laemmli U.K. 1970. Cleavage of stuctural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.

    Google Scholar 

  • Lang M.M., Ingham S.C. and Ingham B.H. 2001. Differentiation of Enterococcus spp. by cell membrane fatty acid methyl ester profiling, biotyping and ribotyping. Lett. Appl. Microbiol. 33: 65-70.

    Google Scholar 

  • Lányí B. 1987. Classical and rapid identification methods for medically important bacteria. Method Microbiol. 19: 1-67.

    Google Scholar 

  • Litopoulou-Tzanetaki E. and Tzanetakis N. 1992. Microbiology of white brined cheese made from raw goat milk. Food Microbiol. 9: 13-19.

    Google Scholar 

  • Lopes M.F.S., Pereira C.I., Rodrigues F.M.S., Martins M.P., Mimoso M.C., Barros T.C., Figueiredo Marques J.J., Tenreiro R.P., Almeida J.S. and Barreto Crespo M.T. 1999. Registered designation of origin areas of fermented food products defined by microbial phenotypes and artificial neural networks. Appl. Environ. Microb. 65: 4484-4489.

    Google Scholar 

  • Macedo A.C., Malcata F.X. and Hogg T.A. 1995. Microbiological 79: 1-11.

    Google Scholar 

  • Manero A. and Blanch A.R. 1999. Identification of Enterococcus spp. with a Biochemical Key. Appl. Environ. Microb. 65: 4425-4430.

    Google Scholar 

  • Massol-Deya A., Odelson D.A., Hickey R.F. and Tiedje J.M. 1995. Bacterial community fingerprinting of amplified 16S and 16-23S ribosomal DNA gene sequences and restriction endonuclease analysis (ARDRA). In: Akkermans A.D.L., van Elsas J.D. and de Bruijn F.J. (eds), Molecular Microbial Ecology Manual, 3.3.2. Kluver Academic Publishers, Dordrecht, The Netherlands, pp. 1-8.

    Google Scholar 

  • Monstein H.-J., Quednau M., Samuelsson A., Ahrné S., Isaksson B. and Jonasson J. 1998. Division of the genus Enterococcus into species groups using PCR-based molecular typing methods. Microbiol. UK 144: 1171-1179.

    Google Scholar 

  • Naïmi A., Beck G. and Branlant C. 1997. Primary and secondary structures of rRNA spacer regions of enterococci. Microbiology 143: 823-824.

    Google Scholar 

  • Naïmi A., Beck G., Monique M., Levébvre G. and Branlanti C. 1999. Determination of the nucleotide sequence of the 23S ribosomal RNA and flanking spacers of an Enterococcus faecium strain, reveals insertion-deletion events in the ribosomal spacer 1 of enterococci. Syst. Appl. Microbiol. 22: 9-21.

    Google Scholar 

  • Normand P., Ponsonnet C., Nesme X., Neyra M. and Simonet P. 1996. ITS analysis of prokaryotes. In: Akkermans A.D.L., van Elsas J.D. and de Bruijn F.J. (eds), Molecular Microbial Ecology Manual, 3.4.5. Kluver Academic Publishers, Dordrecht, The Netherlands, pp. 1-12.

    Google Scholar 

  • Ordoñez J.A., Barneto R. and Ramos M. 1978. Studies on Manchego cheese ripened in olive oil. Milchwissenschaft 33: 609-612.

    Google Scholar 

  • Patel R., Piper K.E., Rouse M.S., Steckelberg J.M., Uhl J.R., Kohner P., Hopkings M.K., Cockerill III F.R. and Kline B.C. 1998. Determination of 16S rRNA sequences of enterococci and aplication to species identification of nonmotile Enterococcus gallinarum isolates. J. Clin. Microbiol. 36: 3399-3407.

    Google Scholar 

  • Pitcher D.G., Saunders N.A. and Owen R.J. 1989. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol. 8: 151-156.

    Google Scholar 

  • Pot B., Vandamme P. and Kersters K. 1994. Analysis of electrophoretic whole-organism protein fingerprints. In: Goodfellow M. (eds), Chemical Methods in Prokaryotic Systematics. Wiley, Chichester, UK, pp. 493-521.

    Google Scholar 

  • Pryce T.M., Wilson R.D. and Kulski J.K. 1999. Identification of enterococci by ribotyping with horseradish-peroxidase labelled 16S rDNA probes. J. Microbiol. Methods 36: 147-155.

    Google Scholar 

  • Sambrook J., Fritsch E.F. and Maniatis T. 1989. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, USA.

    Google Scholar 

  • Schleifer K.M. and Kilpper-Bolz R. 1987. Molecular and chemotaxonomic approaches to the classification of streptococci, enterococci and lactococci: a review. Syst. Appl. Microbiol. 10: 1-19.

    Google Scholar 

  • Sneath, P.H.A. and Johnson R. 1972. The influence on numerical taxonomic similarities of errors in microbiological tests. J. Gen. Microbiol. 73: 377-392.

    Google Scholar 

  • Stiles M.E. and Holzapfel W.H. 1997. Lactic acid bacteria of foods and their current taxonomy. Int. J. Food Microbiol. 36: 1-29.

    Google Scholar 

  • Suzzi G., Caruso M., Gardini F., Lombardi A., Vannini L., Guerzoni M.E., Andrighetto C. and Lanorte M.T. 2000. A survey of the enterococci isolated from an artisanal italian goat's cheese (semicotto caprino). J. Appl. Microbiol. 89: 267-274.

    Google Scholar 

  • Svec P., Devriese, L.A., Sedlacek I., Baele M., Vancanneyt M., Haesebrouck F., Swings J. and Doskar J. 2001. Enterococcus haemoperoxidus sp. nov. and Enterococcus moraviensis sp. nov., isolated from water. Int. J. Syst. Evol. Microbiol. 51: 1567-1574.

    Google Scholar 

  • Teixeira L.M., Facklam R.R., Steigerwalt A.G., Pigott N.E., Merquior V.L.C. and Brenner D.J. 1995. Correlation between phenotypic characteristics and DNA relatedness within Enterococcus faecium strains. J. Clin. Microbiol. 33: 1520-1523.

    Google Scholar 

  • Teixeira L.M., Merquior V.L.C., Vianni M.C.E., Carvalho M.G.S., Fracalanzza S.E.L., Steigerwalt A.G., Brenner D.J. and Facklam R.R. 1996. Phenotypic and Genotypic Characterization of atypical Lactococcus garviae strains isolated from water buffalos with subclinical mastitis and confirmation of L. garviae as a senior subjective synonym of Enterococcus seriolicida. Int. J. Syst. Bacteriol. 46: 664-668.

    Google Scholar 

  • Trovatelli L.D. and Schiesser A. 1987. Identification and significance of enterococci in hard cheese made from raw cow and sheep milk. Milchwissenschaft 42: 717-719.

    Google Scholar 

  • Tyrrell G.J. Bethune R.N., Willey B. and Low D.E. 1997. Species identification of enterococci via intergenic ribosomal PCR. J. Clin. Microbiol. 35: 1054-1060.

    Google Scholar 

  • Vancanneyt M., Snauwaert C., Cleenwerck I., Baele M., Descheemaeker P., Goossens H., Pot B., Vandamme P., Swings J., Haesebrouck F. and Devriese L.A. 2001. Enterococcus villorum sp. nov., an enteroadherent bacterium associated with diarrhoea in piglets. Int. J. Sys. Evol. Microbiol. 51: 393-400.

    Google Scholar 

  • Vandamme P., Pot B., Gillis M., de Vos P., Kersters K. and Swings J. 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol. Rev. 60: 407-438.

    Google Scholar 

  • Wessels D., Jooste P.J. and Mustert J.F. 1990. Technologically important characteristics of Enterococcus isolates from milk and dairy products. Int. J. Food Microbiol. 10: 349-352.

    Google Scholar 

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Authors and Affiliations

  1. Instituto de Biologia Experimental e Tecnológica (IBET) and Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Apartado 12, 2781-901, Oeiras, Portugal

    Paula Isabel Alves, José Joaquim Figueiredo Marques & Maria Teresa Barreto Crespo

  2. Estação Agronómica Nacional, Instituto Nacional de Investigação Agrária e das Pescas, Quinta do Marquês, 2784-505, Oeiras, Portugal

    Maria Paula Martins & José Joaquim Figueiredo Marques

  3. Departamento de Biologia Vegetal e Centro de Genética e Biologia Molecular, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisboa, Portugal

    Teresa Semedo & Rogério Tenreiro

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  1. Paula Isabel Alves
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Alves, P.I., Martins, M.P., Semedo, T. et al. Comparison of phenotypic and genotypic taxonomic methods for the identification of dairy enterococci. Antonie Van Leeuwenhoek 85, 237–252 (2004). https://doi.org/10.1023/B:ANTO.0000020352.29496.4e

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