Enterococcus faecium and Enterococcus durans isolated from cheese: Survival in the presence of medications under simulated gastrointestinal conditions and adhesion properties

In this study, we evaluated the survival of Enterococcus faecium and Enterococcus durans, isolated from cheese, in the presence of medications and under simulated in vitro gastrointestinal conditions. The presence of genes encoding virulence factors, the susceptibility to antimicrobial agents, and adhesion properties were also assessed. Enterococcus faecium and E. durans both exhibited resistance to most of the tested medications but showed a large sensitivity to analgesics and antihypertensives; they also showed wide susceptibility to antimicrobial agents. Enterococcus durans SJRP29 had greater resistance to the presence of medications in comparison with the probiotic Lactobacillus acidophilus La-5. The strains, except for E. durans SJRP05, did not harbor virulence genes. Enterococcus durans SJRP14, SJRP17, and SJRP26 were sensitive to all tested antimicrobial agents. Enterococcus faecium was more stable during the simulation of gastrointestinal tract and showed greater viability. At the end of the assay, except for E. durans SJRP17, all strains showed high viability (>7 log cfu/mL). Enterococcus durans SJRP29 stood out from the other strains and was selected for further evaluation; it tolerated up to 3.0% NaCl at 30 and 37°C, besides having good adhesion properties (high values of auto-aggregation, co-aggregation, and hydrophobicity). Additionally, the microorganism did not show bile salt hydrolase activity or mucin degradation. These results encourage carrying out additional tests to evaluate the probiotic features by using in vitro dynamic models and in vivo tests before applying these strains to a food system.     

Antibiotic susceptibility patterns of Enterococcus strains isolated from Turkish Tulum cheese

The aim of this study was to detect the plasmid profiles, haemolytic activity and antibiotic susceptibility patterns of 47 Enterococcus strains isolated from Turkish Tulum cheese. Enterococcus strains were found to comprise 1–9 plasmids with molecular weight from 2.0 to 47.6 kb. None of the Enterococcus strains displayed haemolytic activity. The Enterococcus strains were found mostly resistant to tetracycline (30 μg) followed by high‐level streptomycin (300 μg). The Enterococcus faecalis strains were found to be highly resistant to antibiotics than Enterococcus faecium and Enterococcus durans strains. In total, 4.3% of the strains exhibited multiple antibiotic resistance patterns.     

Assessment of probiotic characteristics of lactic acid bacteria isolated from fermented yak milk products of Sikkim,India: Chhurpi,Shyow, and Khachu

The present study documents the probiotic attributes of indigenous lactic acid bacteria (LAB) isolated from local fermented Yak milk products namely Chhurpi, Shyow and Khachu prepared in the northern and eastern region of Sikkim in the Himalayas. Samples were collected aseptically and a total of 170 LAB was isolated and screened for putative probiotic properties like hypocholesteromic effect, acid tolerance, bile tolerance, bile salt hydrolase (BSH) activity and cell surface hydrophobicity. It was observed that 70 LAB isolates showed cholesterol lowering activity, out of which 35 isolates were selected that showed 50% and less cholesterol reducing effect in vitro. Acid tolerance test revealed good tolerance of 12 isolates at pH 2.5 and pH 2.0 for up to 2 hours. The tolerance to 0.5% and 1% of three bile salts acid revealed more growth in MRS broth containing taurocholic acid with the isolates revealing good BSH activity leading to bile acid deconjugation. The cell surface hydrophobicity ranged from 20–95%. Furthermore, 16S rRNA gene sequencing revealed Lactobacillus plantarum YD5S and YD9S, L. pentosus YD8S, L. paraplantarum YD11S, Enterococcus lactis YHC20 and E. faecium YY1 as the best isolates with technological properties. The isolates may serve as potential probiotic candidates with potential for hypocholesteromic benefits in the future.     

Safety Assessment and Probiotic Evaluation of Enterococcus Faecium YF5 Isolated from Sourdough

Enterococcus faecium YF5, a strain previously isolated from sourdough, was assessed for safety and probiotic potential. Its virulence and antibiotic resistant phenotypes (cytolysin and gelatinase production, antibiotic susceptibility) and genes (cylA, gelE, ace, agg, esp, and vanA) were surveyed. Results indicated that the tested virulence determinants were nontoxic. In addition, E. faecium YF5 was sensitive to 3 antibiotics such as amoxicillin, vancomycin, and chloramphenicol. Furthermore, results of in vivo animal acute oral toxicity of E. faecium YF5 studies were similar to the control group that indicated no abnormalities. In addition, E. faecium YF5 stably survived in low pH, bile salts, gastric, and intestinal fluids in vitro. Moreover, E. faecium YF5 was found to adhere to human colon cancer cell line HT‐29 at 3.39 (±0.67) × 10⁵ CFU/mL. When cocultured with pathogenic organisms (Enterobacter sakazakii CMCC45402, Escherichia coli CMCC44102, enterohemorrhage Escherichia coli O157: H7 CMCC44828, Salmonella Typhimurium CMCC50071, Shigella flexneri 301, and Shigella sonnei ATCC 29930) and 2 gram‐positive strains (Listeria monocytogenes CMCC54001 and Staphylococcus aureus CMCC 26003), it inhibited these foodborne pathogens with exception of S. aureus. Therefore, E. faecium YF5 can be regarded as a safe strain and it may be used as a probiotic preparation or for microecologics.     

Properties and safety aspects of Enterococcus faecium strains isolated from Chungkukjang, a fermented soy product

Enterococcus faecium isolated from Chungkukjang, a Korean traditional fermented soybean food was studied for their functional characteristics as potential new starter culture and safety. Microbiological analysis of ripened Chungkukjang revealed the presence of an enterococcal population in numbers of up to 6 log CFU per g. Seven isolates with higher activity were selected for further study and the strains were identified as E. faecium. The E. faecium strains showed resistance against simulated gastrointestinal conditions such as acidic environment and the presence of bile salts. These strains also showed bile salt hydrolase activity but neither hemolytic activity nor virulence determinant such as gelE and efaAfm. All strains were susceptible to glycopeptides and lacked potential as vancomycin-resistant enterococci (VRE). Two strains, S2C10 and S2C11, showed inhibited the viability of Listeria monocytogenes in vitro. The ability was probably due to the production of bacteriocin. The lipase activity influenced the stability, while either acidic condition or high temperature did not play a significant role in the activity of the antimicrobial substances. The strains also produced thermostable listericidal antimicrobial substance. For this reason, the strains could be used as selected starters or protective cultures in soybean fermented food production.     

Phenotypic and Genomic Characterization of Enterococcus Species from Some Nigerian Fermented Foods

The gram-positive Enterococci bacteria are generally used as a starter and probiotic cultures in foods. However, they have emerged as one of the leading causes of nosocomial infections worldwide, and this feature is aggravated by the development of antibiotic resistance. Accurate identification of Enterococci at the species level is an important task in food microbiology. In this study, 144 strains of Enterococcus species were isolated from traditional fermented vegetable condiment and West African soft cheese (wara) with the most predominant species being E. gallinarum (75%) followed by E. faecium (14.5%), E. faecalis (7.6%), and E. casselliflavus (2.8%). The strains isolated were characterized and identified using the polyphasic taxonomy approach. Phenotypically, 108 strains were characterized and identified to be E. gallinarum, 21 strains as E. faecium, 11 strains as E. faecalis, and 4 strains as E. casselliflavus. Thirty representative strains were also subjected to genomic characterization, and the result obtained with the phenotypic approach was confirmed. Therefore, the polyphasic taxonomy approach was successful in the accurate identification of the Enterococcus species isolated.     

Optimization and Partial Purification of Bacteriocins from Enterococcus spp. Indigenous to Pakistan

This study was conducted to optimize bacteriocin producing enterococcal strains isolated from indigenous fermented dairy products of Pakistan. Isolates IJ-06, IJ-21, and IJ-31 were identified as Enterococcus faecium and IJ-11 was identified as Enterococcus faecalis. All of these enterococcal isolates were catalase, gelatinase negative, and non-hemolytic on sterile sheep blood. Bacteriocin production trials confirmed E. faecium IJ-06, IJ- 21, and IJ- 31 as the potential producer of bacteriocin showing antimicrobial activity in cell-free supernatant (CFS). E. faecalis IJ-11 displayed activity after partial purification. Optimization of culture conditions for the production of bacteriocin by the selected strains showed that maximum production was achieved at 35–37°C with 1% inoculum after 16 h of incubation and at pH 7–8. The molecular mass of the partially purified enterocins falls in the range of 4.5–4.7 kDa. These enterocins were close to class IIa of bacteriocins and were highly active against Listeria monocytogenes, Bacillus subtilis, Bacillus cereus and other closely related strains.     

Bile salt deconjugation activity of Propionibacterium strains and their cholesterol co‐precipitation abilities

The present study investigated the deconjugation of bile salts and co‐precipitation of cholesterol with deconjugated bile salts by seven Propionibacterium spp. strains in vitro. Propionibacterium spp. could deconjugate sodium glycocholate (1.53–5.31 mM) and sodium taurocholate (0.08–0.25 mM) bile salts. The highest cholesterol precipitation (47.8 µg/mL) was determined with Propionibacterium freudenreichii subsp. freudenreichii SP3 strain in a 0.15% oxgall‐containing medium. Significant (P < 0.05) correlations were observed between cholesterol co‐precipitation and deconjugation of sodium glycocholate among the strains. In vitro bile salt deconjugation activity studies of P. freudenreichii subsp. freudenreichii SP3 strain revealed that this strain may have potential as a probiotic strain for deconjugation of bile salts in vivo studies.     

Antibacterial potential of Enterococcus faecium strains isolated from ewes’ milk and cheese

The study was conducted to evaluate the antibacterial activity of three bacteriocin-producing Enterococcus faecium strains (TW15, TW20 and TW22) isolated from ewes’ milk and cheese sampled in the Patagonian region of Argentina. The strains were tested against spoilage and pathogens microorganisms showing antimicrobial activity towards 4 strains of Listeria monocytogenes, one strain of Listeria innocua and 2 strains of Staphylococcus aureus. E. faecium TW15, E. faecium TW20 and E. faecium TW22 were sensitive to vancomycin. Furthermore, investigation of virulence factors revealed the absence of the genes encoding them. The bacteriocin-like substances (BLISs) produced by the 3 strains were thermostable, pH resistant and can be expressed even in the presence of NaCl (3.0 g/100 g). Moreover, they prove to have a bactericidal mode of action. Results from physicochemical and biochemical characteristics of BLIS produced by these E. faecium strains make them potential candidates to aid in preservation of foods.     

Prevalence,characterization and antibiotic resistance of enterococci from traditional cheeses in Turkey

The aim of the present work is to provide information about Enterococcus strains isolated from traditional Turkish cheese samples in Ankara (Turkey), focusing on their prevalence, phenotypic and genotypic characteristics, and antibiotic resistance. A total of 213 probable enterococcal isolates isolated from 215 samples were identified by phenotypic and genotypic methods. As a result of 16S rDNA sequence analysis, 88 of the 213 enterococci strains were identified as Enterococcus faecium and 125 as Enterococcus faecalis. The E. faecalis strains (58.7%) were identified as the dominant species isolated from cheese samples in Turkey. The 213 Enterococcus strains were tested for susceptibility to 12 different antimicrobial agents. The resistance phenotype were as follow: nalidixic acid (100%), kanamycin (98.6%), rifampicin (78.4%), ampicillin (48.8%), ciprofloxacin (45.5%), erythromycin (18.8%), tetracycline (11.7%), penicillin G (5.6%), chloramphenicol (4.2%), gentamycin (3.8%) and streptomycin (1.4%). None of the strains was resistant to vancomycin. E. faecium strains showed more resistant phenotypes than E. faecalis strains as shown by the antibiotic resistance levels. It was also observed that the resistance of E. faecium and E. faecalis strains against the antibiotics was statistically significant (p ? 0.05). In total, 100% of E. faecium and 88.8% of E. faecalis strains were resistant to multiple drugs.     

Application of bacteriocinogenic Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch in the control of Listeria monocytogenes in fresh Minas cheese

Several strains of Enterococcus spp. are capable of producing bacteriocins with antimicrobial activity against important bacterial pathogens in dairy products. In this study, the bacteriocins produced by two Enterococcus strains (Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch), isolated from cheeses, were characterized and tested for their capability to control growth of Listeria monocytogenes 426 in experimentally contaminated fresh Minas cheese during refrigerated storage. Both strains were active against a variety of pathogenic and non-pathogenic microorganisms and bacteriocin absorption to various L. monocytogenes, Enterococcus faecalis ATCC 19443 and Lactobacillus sakei ATCC 15521 varied according to the strain and the testing conditions (pH, temperature, presence of salts and surfactants). Growth of L. monocytogenes 426 was inhibited in cheeses containing E. mundtii CRL35 up to 12 days at 8 °C, evidencing a bacteriostatic effect. E. faecium ST88Ch was less effective, as the bacteriostatic affect occurred only after 6 days at 8 °C. In cheeses containing nisin (12.5 mg/kg), less than one log reduction was observed. This research underlines the potential application of E. mundtii CRL35 in the control of L. monocytogenes in Minas cheese.     

Probiotic potential and biochemical and technological properties of Lactococcus lactis ssp. lactis strains isolated from raw milk and kefir grains

Lactococcus lactis ssp. lactis is one of the most important starter bacteria used in dairy technology and it is of great economic importance because of its use in the production of dairy products, including cheese, butter, cream, and fermented milks. Numerous studies have evaluated the biochemical and probiotic properties of lactococci; however, limited studies on the probiotic characteristics of lactococci were conducted using strains originating from raw milk and dairy products. Characterizing the probiotic properties of strains isolated from raw milk and fermented milk products is important in terms of selecting starter culture strains for the production of functional dairy products. In this study, biochemical properties (including antibiotic sensitivity, lipolytic activity, amino acid decarboxylation, antioxidant activity) and probiotic properties (including antimicrobial activity, growth in the presence of bile salts, bile salts deconjugation, and hydrophobicity) of 14 Lactococcus lactis strains isolated from raw milk and kefir grains were investigated. Strains originating from kefir grains had better characteristics in terms of antimicrobial activity and bile salt deconjugation, whereas strains from raw milk had better hydrophobicity and antioxidant activity characteristics. None of the strains were able to grow in the presence of bile salt and did not show amino acid decarboxylation or lipolytic activities. Biochemical and probiotic properties of L. lactis strains varied depending on the strain and some of these strains could be used as functional cultures depending on their properties. However, these strains did not possess all of the properties required to meet the definition of a probiotic.     

Survival of biogenic amine‐producing dairy LAB strains at pasteurisation conditions

Biogenic amines (BAs) are low molecular weight organic bases with biological activity, synthesised by the microbial decarboxylation of precursor amino acids such as histidine, tyrosine or arginine. The consumption of food and beverages containing large amounts of BA can have toxicological effects. This paper reports the resistance of aminogenic lactic acid bacteria strains to pasteurisation (63 °C for 30 min). Skimmed milk inoculated with histamine‐ and tyramine‐producing strains of Enterococcus and Lactobacillus was subjected to pasteurisation. The viable number of Enterococcus durans IPLA 655 was reduced by 1 log, while that of Enterococcus faecalis BA64 was reduced by 3 logs. Lactobacillus brevis CECT 3810 did not survive pasteurisation, while the other Lactobacillus strains examined were more resistant; their viable numbers were reduced by about 2 logs. The two most resistant strains, E. durans IPLA 655 and Lactobacillus curvatus VI6, were subjected to higher temperatures. Enterococcus durans was completely inactivated at 78 °C, while some live cells of Lb. curvatus were observed. These results show that some native, metabolically active BA producers could be found in pasteurised milk owing to their resistance to the thermal treatment; their presence could have a negative impact on the final content of BAs in dairy products made from such milk.     

Probiotic and Technological Properties of Enterococci Isolates from Infants and Cheese

Abstract

In the present study 16 enterococcal strains isolated from infant feces and/or Feta cheese were tested for their ability to metabolize in milk and resist specific conditions of the gastrointestinal tract, in order to finally select interesting strains for further studies, for their evaluation as dietary adjuncts. The strains were characterized by phenotypic criteria (API ID 32 STREP) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Whole-cell protein analysis by SDS-PAGE confirmed the taxonomic allocation of 14 strains suggested by phenotypic criteria as E. durans (4 strains), E. faecalis (3 strains), and E. faecium (7 strains). Two strains biochemically characterized as E. faecalis were assigned to other species by SDS-PAGE. All strains grew well in bile concentrations 0–1%. The strains were also able to withstand low pH (3.0) values for 4?h, eventhough for some strains a reduction by ～1–2 log₁₀ cfu/mL was observed. Most of the strains produced tyramine and all of them were susceptible to vancomycin. In addition, six out of seven E. faecium strains from either feces or cheese were β-haemolytic. The strains exhibited antagonistic activities towards enterococci, lactic acid bacteria, and Cl. sporogenes. A considerable phenotypic diversity was found among the test strains concerning their acidifying and proteolytic activities. Results of this study give useful information for the selection of appropriate strains with interesting probiotic and technological properties.     

人粪便来源的屎肠球菌潜在降胆固醇能力研究

高胆固醇血症为心脑血管疾病的重要诱因,使用具有降胆固醇活性的乳酸菌调控血清胆固醇水平具有重要意义。研究从健康人群粪便中分离获得1株动物双歧杆菌和5株屎肠球菌,考察了这些菌株的胆固醇脱除能力、胆盐耐受能力、模拟胃肠道条件耐受能力等特性。结果表明,屎肠球菌MCLF03和MCLF04对胆固醇的脱除能力显著高于其他菌株,脱除率分别为(38.9±3.77)%和(61.0±4.75)%。屎肠球菌MCLF04在0.5%胆盐溶液、人工胃液、人工肠液中的耐受性均高于屎肠球菌MCLF03。屎肠球菌MCLF04具有潜在的降胆固醇特性,可用于降胆固醇功能性食品的开发研究。     

Species distribution,molecular characteristics and vancomycin resistance gene profiles of Enterococcus sp. isolates from farmhouse cheeses in western Turkey

Species distribution, virulence traits and vancomycin resistance gene profiles of Enterococcus isolated from 43 home‐made artisan cheese samples collected from open markets, located in Aydin region of Turkey, were investigated. Of the 129 isolates, 95 were identified as Enterococcus sp.; Enterococcus faecium being the most prevalent species (82.1%), followed by Enterococcus faecalis (13.6%) and Enterococcus durans (1.0%). None of the enterococci were harbouring vanA or vanC, while seven isolates (7.3%) were shown to harbour vanB gene by multiplex PCR. gelE (49.4%) being the most prevalent virulence factor was followed by asa1 (27.3%), esp (22.1%), cylA (4.2%) and hyl (3.1%).     

Evaluation of in vitro probiotic potential of phytase‐producing bacterial strain as a new probiotic candidate

Sixty‐three phytase‐producing bacterial strains were isolated from natural sources, and their probiotic potential was evaluated. Of these, only fifteen strains were selected on the basis of confirmatory plate assay. Among these, five phytase‐producing strains exhibiting potent probiotic properties were identified as Bacillus cereus P1, Bacillus subtilis P6, B. subtilis P7, Pseudomonas aeruginosa P12 and P. aeruginosa P15 on the basis of morphological, biochemical and molecular characteristics. Maximum phytase activity (2.74 EU mL^?1) with potential probiotic properties, that is more than 70% survivability at pH 2.0, up to 2.0% bile salt tolerance, sporulation efficiency of more than 80% and survival in anaerobic condition (94.31%), was revealed by B. subtilis P6 as compared to the well‐established commercial probiotic strains Lactobacillus sporogenes and Lactobacillus casei. Thus, phytase‐producing B. subtilis P6 with promising probiotic features can be used in food and animal feed applications for betterment of mankind after further validation.     

Survival of potentially probiotic enterococci in dairy matrices and in the human gastrointestinal tract

The aim of this study was to investigate the stability of Enterococcus strains isolated from a traditional Portuguese cheese and previously proved to be safe, in dairy matrices, and to assess survival of the best strains in the human gastrointestinal (GI) tract. Enterococcus faecium 32 and Enterococcus durans 37 were added to yoghurt that was ingested by 4 healthy adults. Detection of the enterococcal strains was performed with RAPD-PCR. The intervention trial showed transient colonisation with both strains, via presence in faeces during the ingestion period and disappearance by 10 d post-ingestion. Viable numbers of enterococci increased during the consumption period by 1.8–4.4 log-values, and returned to baseline level during the follow-up period. Based on data of the dairy matrix stability trials and human intervention study involving yoghurt ingestion, E. faecium 32 survived well both in the food matrix and in the human GI tract, thus showing probiotic potential.     

Bile salt deconjugation ability,bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains

Eleven strains of lactobacilli were screened for their bile salt deconjugation ability, bile salt hydrolase activity (BSH) and co-precipitation of cholesterol with deconjugated bile. Bile salt deconjugation as determined by the release of cholic acid showed that more cholic acid was liberated from the deconjugation of sodium glycocholate than sodium taurocholate, and Lactobacillus acidophilus strains had higher deconjugation ability than L. casei strains. BSH activity, as quantified by the amount of taurine or glycine liberated from conjugated bile salts, indicated that substrate specificity was more towards glycine-conjugated bile compared to taurine-conjugated bile. Co-precipitation of cholesterol with cholic acid was observed from deconjugation of both conjugated bile, with more cholesterol being precipitated upon deconjugation of sodium glycocholate than upon that of sodium taurocholate. Cholesterol co-precipitation with deconjugated bile increased with decreasing pH. L. acidophilus ATCC 33200, 4356 and 4962 and L. casei ASCC 1521 showed highest deconjugation ability and BSH activity towards bile mixtures that resemble the human bile, and may be promising candidates to exert beneficial bile deconjugation activity in vivo.     

Assimilation of cholesterol and probiotic characterisation of yeast strains isolated from raw milk and fermented foods

In this study, cholesterol assimilation ratios and some probiotic characteristics of yeasts were investigated. For this purpose, yeasts were isolated from milk and foods that were naturally fermented and not containing starter culture. In vitro cholesterol assimilation properties were determined in media. The Cholesterol assimilation by yeast strains ranged between 1.36 and 73.33%. Twenty‐one yeast strains showing high assimilation percentage were selected, and their acid tolerance, bile tolerance, bile salt deconjugation activity and survival in simulated gastrointestinal conditions were investigated. Among the strains assessed, 12 of them showed probiotic characteristics.