Phenotypic and genetic diversity of enterococci isolated from Italian cheeses

Veneto Agricoltura-Istituto per la Qualità e le Tecnologie Agroalimentari, Thiene (VI), Italy.
J Dairy Res (Impact Factor: 1.6). 06/2001; 68(2):303-16.
Source: PubMed


In the present study, 124 enterococcal strains, isolated from traditional Italian cow, goat and buffalo cheeses, were characterized using phenotypic features and randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). The RAPD-PCR profiles obtained with four primers and five different amplification conditions were compared by numerical analysis and allowed an inter- and intraspecific differentiation of the isolates. Whole cell protein analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used as a reference method for species identification. The strains were identified as Enterococcus faecalis (82 strains), E. faecium (27 strains), E. durans (nine strains), E. gallinarum (four strains) and E. hirae (two strains). Species recognition by means of RAPD-PCR was in agreement with the SDS-PAGE results except for eight strains of E. faecium that clustered in separated groups. On the other hand, phenotypic identification based on carbohydrate fermentation profiles, using the rapid ID 32 STREP galleries, gave different results from SDS-PAGE in 12.1% of the cases. The majority of the strains had weak acidifying and proteolytic activities in milk. One E. faecium strain showed vanA (vancomycin resistance) genotype while four strains showed a beta-haemolytic reaction on human blood. Several strains showed antagonistic activity towards indicator strains of Listeria innocua, Clostridium tyrobutyricam and Propionibacterium freudenreichii subsp. shermanii.

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    • "Antibiotic susceptibility of enterococcal isolates the production of various volatile compounds, such as acetaldehyde, acetoin, diacetyl, and ethanol, which are responsible for the formation of the unique aroma and flavor of the final product (Andrighetto et al., 2001; Sarantinopoulos et al., 2001; Giraffa, 2002; Abeijón et al., 2006; Foulquié- Moreno et al., 2006). Due to their interesting metabolic and biotechnological traits (the ability to metabolize citrate, proteolytic and esterolytic activities, bacteriocin production, and their probiotic characteristics), enterococci may be utilized in food fermentation as commercial starter cultures (Tsakalidou et al., 1993; Centeno et al., 1999; Giraffa, 2003; Menéndez et al., 2004). "
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    ABSTRACT: Enterococci represent the most controversial group of dairy bacteria. They are found to be the main constituent of many traditional Mediterranean dairy products and contribute to their characteristic taste and flavor. On the other hand, during the last 50 years antibiotic-resistant enterococci have emerged as leading causes of nosocomial infections worldwide. The aim of this study was to determine the diversity, technological properties, antibiotic susceptibility and virulence traits of 636 enterococci previously isolated from 55 artisan dairy products from 12 locations in the Western Balkan countries (WBC) of Serbia, Croatia and Bosnia and Herzegovina. All strains were identified both by microbiological and molecular methods. The predominant species was Enterococcus durans, followed by Enterococcus faecalis and Enterococcus faecium. Over 44% of the isolates were resistant to ciprofloxacin and erythromycin, while 26.2% of the isolates were multi-resistant to three or more antibiotics belonging to different families. 185 isolates (29.1%) were susceptible to all 13 of the antibiotics tested. The antibiotic-susceptible isolates were further tested for possible virulence genes and the production of biogenic amines. Finally, five enterococci isolates were found to be antibiotic susceptible with good technological characteristics and without virulence traits or the ability to produce biogenic amines, making them possible candidates for biotechnological application as starter cultures in the dairy industry.
    Frontiers in Microbiology 09/2015; 6. DOI:10.3389/fmicb.2015.00954 · 3.99 Impact Factor
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    • "RAPD-PCR was carried out using the primer M13 (5 -GAG GGT GGC GGT TCT-3 ) (Huey and Hall, 1989). PCR reactions and amplification conditions were performed as described by Andrighetto et al. (2001) and Suzzi et al. (2000). Rep-PCR analysis was carried out using the primer GTG 5 (5 -GTG GTG GTG GTG GTG-3 ) and amplification conditions as previously described by Gevers et al. (2001). "
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    ABSTRACT: A total of 104 lactic acid bacteria were isolated from ogi, wara, fermenting cassava mash for local food product gari and raw milk for nono. They were characterized phenotypically and divided into six main groups, which are facultative heterofermentative rods, obligate heterofermentative rods, tetrad-forming homofermentative cocci, homofermentative cocci, heterofermentative cocci, and an unidentified group. A total of 40 strains with good acidification, hydrogen peroxide production, and fermentation of indigestible sugar such as raffinose were selected. They were further characterized employing genotypic fingerprinting techniques, such as RAPD-PCR with primer M13, rep-PCR with primer (GTG)5 and sequencing of the 16S rDNA genes for starter culture development. RAPD-PCR did not give accurate delineation of the strains, but with the combination of rep-PCR fingerprinting and 16S rDNA sequencing, representative strains from rep-PCR clusters at 80% Pearson's correlation coefficient (r) grouped the organisms to nine (9) clusters. These were identified as Lactobacillus fermentum (7), Lactobacillus plantarum (6), Lactobacillus pentosus (2), Pediococcus pentosaceus (10), Pediococcus acidilactici (4), Enterococcus faecium (7), and the unidentified Lactobacillus species (4) were assigned into Lactobacillus fermentum group. A polyphasic taxonomic approach for characterization proved useful in the accurate typing of the LAB strains.
    Food Biotechnology 04/2012; 26(2):124-142. DOI:10.1080/08905436.2012.670831 · 0.51 Impact Factor
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    • "However, enterococci with proven pathogenic potential, both at the phenotypic and genotypic level, were also isolated from food sources (e.g. different cheeses) [24]. A recent secretome analysis of E. faecalis JH2-2, a culture collection strain, revealed the presence of various pathogenicity-associated traits, including coccolysin and proteins such as elongation factor Tu, chaperone DnaK and glycolytic enzymes, which might exhibit a second function, e.g. "
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    ABSTRACT: The use of Enterococcus faecalis in the food industry has come under dispute because of the pathogenic potential of some strains of this species. In this study, we have compared the secretome and whole-cell proteome of one food isolate (E. faecalis DISAV 1022) and one clinical isolate (E. faecalis H1) by 2-DE and iTRAQ analyses, respectively. Extracellular protein patterns differed significantly, with only seven proteins common to both strains. Notably, only the clinical isolate expressed various well-characterized virulence factors such as the gelatinase coccolysin (GelE) and the extracellular serine proteinase V8 (SprE). Moreover, various other putative virulence factors, e.g. superoxide dismutase, choline- and chitin-binding proteins and potential moonlighting proteins, have been detected exclusively in the secretome of the clinical isolate, but not in the food isolate. The iTRAQ analysis of whole-cell proteins of the two strains highlighted a stronger expression of pathogenic traits such as an endocarditis-specific antigen and an adhesion lipoprotein in the pathogenic strain E. faecalis H1. Subsequently, six food isolates (including E. faecalis DISAV 1022) and six clinical isolates (including E. faecalis H1) were tested for the presence of gelatinase and protease activity in the culture supernatants. Both enzymatic activities were found in the clinical as well as the food isolates which clearly indicates that protease expression is strain specific and not representative for pathogenic isolates. Genetic analyses revealed that not only the gelatinase and serine protease genes but also the regulatory fsr genes must be present to allow protease expression.
    Proteomics 02/2012; 12(3):431-47. DOI:10.1002/pmic.201100468 · 3.81 Impact Factor
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