Identification and characterization of enterococci from bryndza cheese

AIMS: To identify enterococci isolated from sheep milk cheese--bryndza, and to compare differences in the composition of enterococcal microflora affected by the season, and to evaluate the potential presence of vancomycin resistance and virulence determinants. METHODS AND RESULTS: Bacterial strains were isolated during analysis of bryndza cheese and identified on the genus and species level by phenotypic methods and with commercial biochemical sets. The identification of the species, Enterococcus faecium, Ent. durans and Ent. faecalis, was confirmed by PCR using species-specific primers for ddl genes. PCR was also used for assessment of presence of vanA and vanB genes and virulence determinants gelE, agg and cytolysin genes namely: cylL(L), cylL(S), cylM, cylB and cylA. Among 308 Enterococcus sp. strains, 177 isolates were proved to be Ent. faecium, 59 to be Ent. durans and 41 to be Ent. faecalis. Vancomycin resistance genes vanA and vanB were not detected. Agar plate testing confirmed their absence. Gene gelE, however, was found in 20 Ent. faecalis isolates, but only 13 of them showed gelatinase-positive phenotype. Seven isolates had five cytolysin genes, but none of the isolates exhibited a positive haemolytic phenotype. Four isolates possessed the agg gene. The prevalence of Ent. faecium species was highest in samples from the winter season harvest. CONCLUSIONS: Ent. faecium is the dominant enterococcal species in bryndza cheese and the most prevalent in the winter season product. None of the Enterococcus sp. strains was proved to have vanA or vanB genes and the vancomycin resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of enterococcal microflora in bryndza cheese and its evaluation for the presence of vanA and vanB genes as well as virulence determinants.     

Characterization of 'faecal streptococci' as indicators of faecal pollution and distribution in the environment

The recent revision of the taxonomy of 'faecal streptococci' prompted us to verify the importance of identifying the species of this group of cocci. During a study carried out to assess the hygienic quality of environmental samples from a variety of sources, we isolated 198 strains named faecal streptococci on the basis of conventional international tests (EVA broth multiple tube test) used for Public Health purposes. The predominant species were Enterococcus faecalis (39%) and Ent. faecium (29%), followed by Ent. durans/hirae, Ent. casseliflavus/gallinarum, Ent. raffinosus, with a different prevalence of the species depending on the source. Eighty-four per cent of isolates were true faecal species. Only one isolate was identified as belonging to the Streptococcus genus. The authors stress the opportunity to identify the species. This may help to clarify the ecological and epidemiological characteristics of intestinal enterococci and streptococci in the environment, in drinking and recreational waters and their meaning as indicators of faecal pollution. All isolates were tested for their susceptibility to some antimicrobial agents widely used in medical therapy and the pattern was compared with the pattern of isolates from clinical specimens.     

Molecular identification and diversity of enterococci isolated from Slovak Bryndza cheese

Three hundred and eight presumed enterococcal isolates were recovered from Bryndza, a soft sheep milk cheese. The cheese samples were obtained from five different commercial distributors in Slovakia and were taken at three different seasonal intervals. All isolates were identified to the species level using genotypic tools. Species-specific PCR using ddl genes highlighted the predominance of Enterococcus faecium (176 isolates) and assigned 50 isolates to the species Enterococcus faecalis. The remaining 82 isolates were classified using repetitive element sequence-based polymerase chain reaction (PCR) with primer (GTG)(5)-(GTG)(5)-PCR, in combination with phenylalanyl-tRNA synthase gene (pheS) sequence analysis and by whole-cell protein analysis (SDS-PAGE). These strains were identified as Enterococcus durans (59 strains), Enterococcus italicus (8 strains), Enterococcus casseliflavus (3 strains), Enterococcus gallinarum (3 strains), Enterococcus hirae (1 strain), and 8 strains were members of the species Lactococcus lactis. Of the seven enterococcal species isolated, three of them, E. durans, E. faecalis and E. faecium were present in all samples studied, with E. faecium as the predominant one. The precise identification of enterococci in Bryndza cheese is an essential step in the process of evaluation of their functional properties which will be further studied and assessed.     

Population structure and safety aspects of Enterococcus strains isolated from artisanal dry fermented sausages produced in Argentina

Enterococci population from Argentinean artisanal dry fermented sausage was identified and their safety aspects were evaluated. Species-specific PCR was used to distinguish between Enterococcus faecium (56%) and Enterococcus faecalis (17%). Other isolates (27%) were identified as Enterococcus durans , Enterococcus casseliflavus and Enterococcus mundtii by using 16S RNA gene sequence. RAPD analyses showed different biotypes for Ent. faecium and Ent. faecalis species. Low incidence of antibiotic resistance and high virulence traits in Ent. casseliflavus and Ent. faecalis were found; the majority of the Ent. faecium strains were shown to be free of virulence factors. The absence of virulence/resistance traits and the anti-Listeria activity of Ent. faecium isolates may be exploited to enhance natural preservation thereby guaranteeing organoleptic/safety characteristics of artisanal fermented sausages.     

Composition of the enterococcal and streptococcal intestinal flora of poultry

Identification to species level showed that Enterococcus faecalis and Ent. faecium largely dominated the enterococcal and streptococcal gut flora of 1-d-old chicks. Enterococcus faecalis was rare in 3- to 5-week-old broilers. Two species, Ent. faecium and Streptococcus alactolyticus , were isolated from nearly all broilers examined. Enterococcus hirae and Ent. durans were found in the small intestines of this category of poultry.
In layers and parent stock of over 12 weeks of age, Ent. cecorum dominated with Strep. alactolyticus ranking next. Other species were isolated irregularly. Enterococcus avium and Ent. gallinarum , originally described from chickens, were rarely found. These species did not appear to belong to the normal intestinal flora of poultry.     

Composition of the enterococcal and streptococcal intestinal flora of poultry

Identification to species level showed that Enterococcus faecalis and Ent. faecium largely dominated the enterococcal and streptococcal gut flora of 1-d-old chicks. Enterococcus faecalis was rare in 3- to 5-week-old broilers. Two species, Ent. faecium and Streptococcus alactolyticus, were isolated from nearly all broilers examined. Enterococcus hirae and Ent. durans were found in the small intestines of this category of poultry. In layers and parent stock of over 12 weeks of age, Ent. cecorum dominated with Strep. alactolyticus ranking next. Other species were isolated irregularly. Enterococcus avium and Ent. gallinarum, originally described from chickens, were rarely found. These species did not appear to belong to the normal intestinal flora of poultry.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent.faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium -like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum , shared characteristics with Ent. columbae.
The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol.
Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.     

The effect of ampicillin and tylosin on the faecal enterococci of healthy young chickens

Enterococcal isolates from young chickens were differentiated into one of three species, namely Enterococcus faecalis, Ent. faecium and Ent. gallinarum . The proportion of each species among the enterococcal population changed with time in birds not dosed with antibiotics. This pattern of change was modified in birds dosed with either tylosin or ampicillin even though ampicillin did not select for ampicillin resistance among the enterococcal population. A gradual increase in tylosin resistance was recorded with time among the enterococci of the 'undosed' control birds. This was associated with an increase in the proportion of Ent. faecium , a species commonly resistant to tylosin, among the enterococci of the birds as they grew older.     

The effect of ampicillin and tylosin on the faecal enterococci of healthy young chickens

Enterococcal isolates from young chickens were differentiated into one of three species, namely Enterococcus faecalis, Ent. faecium and Ent. gallinarum. The proportion of each species among the enterococcal population changed with time in birds not dosed with antibiotics. This pattern of change was modified in birds dosed with either tylosin or ampicillin even though ampicillin did not select for ampicillin resistance among the enterococcal population. A gradual increase in tylosin resistance was recorded with time among the enterococci of the 'undosed' control birds. This was associated with an increase in the proportion of Ent. faecium, a species commonly resistant to tylosin, among the enterococci of the birds as they grew older.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats.

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent. faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium-like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum, shared characteristics with Ent. columbae. The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol. Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.     

Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture andripening of Manchego cheese

The inhibitory effect of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4, on Listeria monocytogenes strains Ohio and Scott A during themanufacture and ripening of Manchego cheese was investigated. Raw ewe's milk wasinoculated with ca 10⁵ cfu ml⁻¹ of L.monocytogenes and with 1% of a commercial lactic starter, 1% of an Ent. faecalis INIA 4 culture, or 1% of each culture. Manchego cheeses were manufactured according tousual procedures. Listeria monocytogenes Ohio counts decreased by 3 log units after8 h and by 6 log units after 7 d in cheese made from milk inoculated with Ent. faecalis INIA 4 or with both cultures, whereas no inhibition was recorded after 60 d in cheese made frommilk inoculated with commercial lactic starter. Listeria monocytogenes Scott A wasnot inhibited by enterocin 4 during cheese manufacture, but decreases of 1 log unit after 7 d andof 2 log units after 60 d were achieved in cheese made from milk inoculated with bothcommercial lactic starter and Ent. faecalis INIA 4.     

Production of bacteriolytic enzymes as a tool for characterizing enterococci

Bacteriolytic enzymes secreted by log-phase cultures of enterococci ( Enterococcus faecalis, Ent. faecium, Ent. durans, Ent. hirae, Ent. casseliflavus, Ent. avium, Ent. mundtii ) were analysed by means of a zymogram technique to resolve activities according to the size of their polypeptide component and their specificities towards different substrates. Heterogeneous patterns of lytic activity were observed with different species. For each test substrate, homogeneous patterns of lytic activities were observed with strains of the same species, except for Ent. faecalis strains, which showed heterogeneous lytic patterns even towards the same substrate, and could be divided into at least four different groups according to their lytic pattern. No lytic activity was common to all strains tested. Results of zymogram analysis of Enterococcus bacteriolytic enzymes were consistent with current knowledge on enterococcal taxonomy, indicating that this analytical approach may be a useful tool for fine-tuned characterization of different enterococcal strains.     

Biogenic amine-forming microbial communities in cheese

The aim of this study was to screen two cheese starter cultures and cheese-borne microbial communities with the potential to produce biogenic amines in cheese during ripening. Bacteria of the genera Enterococcus and Lactobacillus and coliform bacteria were isolated from Dutch-type semi-hard cheese at the beginning of the ripening period. Statistically significant counts of bacterial isolates were screened for the presence of specific DNA sequences coding for tyrosine decarboxylase (tyrDC) and histidine decarboxylase (hDC) enzymes. The PCR analysis of DNA from 14 Enterococcus and 3 Lactobacillus isolates confirmed the presence of the targetted DNA sequences. Simultaneously, 13 tyrDC- and 3 hDC-positive isolates were grown in decarboxylase screening medium and this was followed by HPLC analysis of the produced tyramine and histamine. Conventional and molecular taxonomic analyses of the above-mentioned isolates identified the following species: Enterococcus durans (7 strains), Enterococcus faecalis (3 strains), Enterococcus faecium (1 strain), Enterococcus casseliflavus (3 strains), Lactobacillus curvatus (1 strain), Lactobacillus lactis (1 strain) and Lactobacillus helveticus (1 strain). All of the above Enterococcus and two of the Lactobacillus strains originated from contaminating microbial communities. The L. helveticus strain, which was tyrosine decarboxylase-positive and exhibited tyramine production, originated from starter culture 1 used for cheese production. Comparison of partial tyrDC sequences of positive Enterococcus isolates revealed 89% sequence similarity, and that of hDC-positive Lactobacillus isolates revealed 99% sequence similarity.     

Evaluation of a biochemical test scheme for identifying clinical isolates of Enterococcus faecalis and Enterococcus faecium

AIMS: To evaluate the full test scheme of Facklam and Sahm (1995) for the identification of clinical enterococcal isolates to genus and species level. METHODS AND RESULTS: Fifty-nine clinical isolates, previously provisionally classed as enterococci on the basis of just four biochemical tests of Facklam and Sahm and one other test, were subjected to genus and species identification using the full identification scheme of Facklam and Sahm; 98% of these strains were confirmed to be enterococci and of these, 69% were identified as Enterococcus faecalis and 31% as Enterococcus faecium. Six tests in the scheme (out of 24) gave anomalous or unreliable results for some strains, and two gave unexpected results for the majority of strains presumptively identified as Ent. faecium. CONCLUSIONS: Nine (out of 12) genus tests and nine (out of 12) species tests from the Facklam and Sahm scheme were reliable. Testing for the presence of the Lancefield antigen D was also useful. The majority of presumptive Ent. faecium strains gave different results for the sorbitol and raffinose tests from that expected. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates the level of reliability for each of the tests in a current enterococcal identification scheme for differentiating clinical isolates, and showed that two tests gave consistently different test results from those expected for Ent. faecium.     

Diversity and dynamics of lactobacilli populations during ripening of RDO Camembert cheese

The diversity and dynamics of Lactobacillus populations in traditional raw milk Camembert cheese were monitored throughout the manufacturing process in 3 dairies. Culture-dependent analysis was carried out on isolates grown on acidified de Man - Rogosa - Sharpe agar and Lactobacillus anaerobic de Man Rogosa Sharpe agar supplemented with vancomycin and bromocresol green media. The isolates were identified by polymerase chain reaction - temperature gradient gel electrophoresis (PCR-TGGE) and (or) species-specific PCR and (or) sequencing, and Lactobacillus paracasei and Lactobacillus plantarum isolates were characterized by pulsed field gel electrophoresis (PFGE). Milk and cheese were subjected to culture-independent analysis by PCR-TGGE. Presumed lactobacilli were detected by plate counts throughout the ripening process. However, molecular analysis of total DNA and DNA of isolates failed to detect Lactobacillus spp. in certain cases. The dominant species in the 3 dairies was L. paracasei. PFGE analysis revealed 21 different profiles among 39 L. paracasei isolates. Lactobacillus plantarum was the second most isolated species, but it occurred nearly exclusively in one dairy. The other species isolated were Lactobacillus parabuchneri, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus helveticus, a Lactobacillus psittaci/delbrueckii subsp. bulgaricus/gallinarum/crispatus group, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, Lactobacillus brevis, Lactobacillus kefiri, and Lactobacillus perolens. Lactobacilli diversity at the strain level was high. Dynamics varied among dairies, and each cheese exhibited a specific picture of species and strains.     

Glutamate dehydrogenase activity in lactobacilli and the use of glutamate dehydrogenase-producing adjunct Lactobacillus spp. cultures in the manufacture of cheddar cheese

AIMS: The study was undertaken to investigate the occurrence of glutamate dehydrogenase activity in different species of lactobacilli, and to determine, in a series of cheese-making trials, the effects of glutamate dehydrogenase-producing adjunct cultures on sensory attribute development during the maturation of cheddar cheese. METHODS AND RESULTS: The presence of dehydrogenase activity with glutamate as substrate was monitored in cell lysates of >100 strains from 30 different species of lactobacilli using a qualitative colorimetric plate screening assay. Activity was detectable in 25 of the 29 representative species obtained from culture collections and in 12 of the 13 non-starter species isolated from cheese. There were pronounced interspecies and strain differences in the occurrence, level and pyridine nucleotide specificity of the glutamate dehydrogenase activity detected. Among the non-starter lactobacilli the highest frequency of enzyme occurrence and activity was detected in the Lactobacillus plantarum isolates. The establishment of glutamate dehydrogenase-producing adjunct strains in the predominant population of lactobacilli in the cheese curd affected the formation of a number of volatile compounds in ripening cheddar cheese, while the presence of Lact. plantarum strains, in particular, was associated with an intensification and acceleration of aroma and flavour development during the maturation period. CONCLUSIONS: Glutamate dehydrogenase formation by lactobacilli is a strain-dependent metabolic attribute, and adjunct cultures expressing the activity that are able to proliferate during cheese ripening have a positive impact on the rate of development and the intensity of cheddar cheese aroma and flavour development. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been demonstrated that some strains of glutamate dehydrogenase-producing lactobacilli have potential use as adjunct cultures to accelerate and intensify aroma and flavour formation during the manufacture of cheddar and, by analogy, other similar varieties of cheese. The importance of phenotypic discriminative monitoring of the dominant lactobacilli present during ripening to confirm adjunct establishment and population complexity was highlighted as was the requirement to establish the metabolic attributes of the non-starter population in uninoculated control cheeses in comparative trials.     

Population dynamics and antagonistic potential of enterococci colonizing the phyllosphere of grasses

AIMS: To investigate the spatial and temporal dynamics of enterococci colonizing forage grass and their ability to produce bacteriocins. Methods and RESULTS: Enterococci could be detected on above-ground plant parts throughout the growing season, with high continuity but low cell numbers (2.60 x 101-6.16 x 104 cfu g-1 fresh matter). A total of 750 strains were isolated and identified by their whole-cell protein patterns as Enterococcus faecalis (7.9%), Ent. mundtii (7.9%), Ent. casseliflavus (5.5%), Ent. faecium (5.2%) and Ent. sulfureus (0.1%). The vast majority of the strains (69.7%) formed a homogeneous 16S rDNA genotype that differed from those of known enterococci. A screening for antagonistic activity using an agar spot test revealed that 18.4% of all isolates were potential antagonists. Partially-purified proteins extracted from cell-free culture supernatant fluids of various species were characterized as pH- and heat-stable bacteriocins active against a wide range of lactic acid bacteria, clostridia and Listeria. The producing strains were antagonistically active even on 'phylloplane agar' at temperatures between 4 and 37 degrees C. CONCLUSION: Enterococci are a common part of the epiphytic microflora of grasses, displaying probably some antagonistic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide new information on the distribution, species diversity and antagonistic potential of enterococci in the phyllosphere.     

Genetic diversity and safety aspects of enterococci from slightly fermented sausages

AIMS: To determine the biodiversity of enterococci from slightly fermented sausages (chorizo and fuet) at species and strain level by molecular typing, while considering their safety aspects. METHODS AND RESULTS: Species-specific PCR and partial sequencing of 16S rRNA and sodA genes were used to identify enterococcal population. Enterococcus faecium was the most frequently isolated species followed by E. faecalis, E. hirae and E. durans. Randomly amplified polymorphic DNA (RAPD)-PCR revealed species-specific clusters and allowed strain typing. Sixty strains of 106 isolates exhibited different RAPD profiles indicating a high genetic variability. All the E. faecalis strains carried virulence genes (efaAfs, esp, agg and gelE) and all E. faecium isolates carried efaAfm gene. Enterococcus faecalis showed higher antibiotic resistance than the other species. Only one E. faecium strain showed vanA genotype (high-level resistance to glycopeptides) and E. gallinarum and E. casseliflavus/flavescens isolates showed vanC1 and vanC2/C3 genotypes (low-level resistance only to vancomycin) respectively. CONCLUSIONS: E. faecalis has been mainly associated with virulence factors and antimicrobial multi-resistance and, although potential risk for human health is low, the presence of this species in slightly fermented sausages should be avoided to obtain high quality products. SIGNIFICANCE AND IMPACT OF THE STUDY: The enterococcal population of slightly fermented sausages has been thoroughly characterized. Several relevant safety aspects have been revealed.     

Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces

A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by alpha-chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS-PAGE, was approximately 3.4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B.