Phenotypic and genetic diversity of enterococci isolated from Italian cheeses.
ABSTRACT 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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ABSTRACT: The aim of this study was to identify and characterize the lactic acid bacteria (LAB) of artisanal Golija raw and cooked cows’ milk cheeses traditionally manufactured without the addition of starter culture. A total of 188 Gram-positive and catalase-negative isolates of Golija cheeses were obtained from seven samples of different ripening time. Phenotypebased assays as well as rep-PCR and 16S rDNA sequence analysis were undertaken for all 188 LAB strains. The most diverse species were isolated from 20-day-old BGGO8 cheese (Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus casei/paracasei, Lactobacillus sucicola, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis bv. diacetylactis, Enterococcus faecium, Enterococcus durans and Leuconostoc mesenteroides). In other Golija cheeses Lactobacillus reuteri, Lactobacillus curvatus, Lactobacillus rhamnosus, Lactococcus lactis subsp. cremoris, Lactococcus garvieae, Streptococcus thermophilus and Leuconostoc pseudomesenteroides were found. Pronounced antimicrobial properties showed enterococci (13/42) and lactococci (12/31), while the good proteolytic activity demonstrated lactococci (13/31) and lactobacilli (10/29).Archives of Biological Sciences 01/2014; 66(1):179-192. · 0.79 Impact Factor
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ABSTRACT: Enterococcus faecalis (n = 834) and Enterococcus faecium (n = 135) from blood and feces of hospitalized humans, from feces of outpatients and livestock and from food were screened for their susceptibility to a quaternary ammonium compound (didecyldimethylammoniumchloride, DDAC) and to 28 antibiotics by micro-/macrodilution. The maximum DDAC-MIC in our field study was 3.5 mg/l, but after adaptation in the laboratory, MIC values of 21.9 mg/l were observed. Strains for which DDAC had MICs > 1.4 mg/l ("non-wildtype," in total: 46 of 969 isolates/4.7%) were most often found in milk and dairy products (14.6%), while their prevalence in livestock was generally low (0-4%). Of human isolates, 2.9-6.8% had a "non-wildtype" phenotype. An association between reduced susceptibility to DDAC, high-level-aminoglycoside resistance and aminopenicillin resistance was seen in E. faecium (p < 0.05). No indications for a common source of non-wildtype strains were found by RAPD-PCR; however, several non-wildtype E. faecalis shared the same variant of the emeA-gene. In addition, bacteria (n = 42) of different genera were isolated from formic acid based boot bath disinfectant (20 ml of 55% formic acid/l). The MICs of this disinfectant exceeded the wildtype MICs up to 20-fold (staphylococci), but were still one to three orders of magnitude below the used concentration of the disinfectant (i. e., 1.1% formic acid). In conclusion, the bacterial susceptibility to disinfectants still seems to be high. Thus, the proper use of disinfectants in livestock surroundings along with a good hygiene praxis should still be highly encouraged. Hints to a link between antibiotic resistance and reduced susceptibility for disinfectants-as seen for E. faecium-should be substantiated in further studies and might be an additional reason to confine the use of antibiotics.Frontiers in Microbiology 01/2014; 5:88. · 3.90 Impact Factor
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ABSTRACT: Milk proteins are precursors of biologically active components that are released by enzymatic proteolysis. Among the biological activities recognised in milk components, the angiotensin-I converting enzyme (ACE)-inhibitory and immunomodulatory activities are of great interest. In the present work the ACE-inhibitory and immunomodulatory activities were analysed in milks fermented by two bacterial strains isolated from Italian dairy products, Enterococcus faecalis TH563 or Lactobacillus delbrueckii subsp. bulgaricus (L. delb. bulgaricus) LA2. ACE-inhibitory activity was determined by an already established enzymatic method and immunomodulatory activity by the proliferation of bovine peripheral blood lymphocytes (BPBL) taken from nine cows. BPBL were incubated for 48 h with increasing concentrations of peptide fractions (< 5000 g·mol−1) extracted from the two fermented milks. Concanavalin A (conA), a known activator of lymphocyte proliferation, was used as a positive control. Fermentation products from E. faecalis TH563 showed a significantly (P < 0.05) greater ACE-inhibitory activity than that obtained by L. delb. bulgaricus LA2 (69.43 ± 3.12% vs. 60.86 ± 1.01%). The immunomodulatory activity showed a large interanimal variability. Peptide fractions from milk fermented by L. delb. bulgaricus LA2 significantly inhibited BPBL proliferation at concentrations of 5, 25 and 50 μg·mL−1 in the presence of conA (P < 0.01). E. faecalis TH563 did not significantly modify BPBL proliferation at any peptide concentration used. In conclusion, L. delb. bulgaricus LA2-fermented milk showed ACE-inhibitory and immunomodulatory activities, while E. faecalis TH563-fermented milk had high ACE-inhibitory activity, suggesting a possible use of these strains for determining bioactive properties in dairy products.Dairy Science and Technology 01/2010; 90(4):469-476. · 1.38 Impact Factor