Genotypic and phenotypic heterogeneity among lactococci isolated from traditional Pecorino Sardo cheese

Twenty-nine Lactococcus lactis isolates from one traditional 24 h-old Pecorino Sardo cheese were characterized phenotypically, technologically and genotypically in order to assess the biodiversity within this wild microbial population. Two DNA-based techniques, plasmid profiling and PFGE, were used for the genetic typing of the isolates. All 29 isolates were characterized at strain level and eight different genotypes were recognized. In addition, by combining the results from plasmid profile analysis and PFGE, it was possible to identify closely related isolates probably belonging to the same clonal lineage. The dominant biotype was identified in the 24 h-old cheese, as were the strains believed to act as starters for the curd. Atypical lactococci, able to grow in 6.5% NaCl, were isolated. The results suggest that wild bacterial populations should be preserved in order to protect the traditional raw milk cheeses, and to select new starter strains for the dairy industry.     

Diversity among lactococci isolated from ewes' raw milk and cheese

P. GAYA, M. BABÍN, M. MEDINA and M. NUÑEZ.1999.The technological and genetic characteristics of lactococci present in ewes' raw milk and 1-d-old ewes' raw milk cheeses sampled over a 1-year period were investigated. The proportion of lactic acid bacteria isolates from milk samples able to decrease milk pH by more than 1·25 units after 6 h incubation at 30 °C reached 14·5% in spring vs 10·7% in summer, 8·3% in autumn and 3·0% in winter. In 1-d-old cheese samples, the proportion of lactic acid bacteria able to lower milk pH by more than 1·25 units increased up to 32·3% in spring vs 23·4% in summer, 8·0% in autumn and 10·3% in winter. Fast acid-producing lactic acid bacteria mainly belonged to the genus Lactococcus . Using polymerase chain reaction protocols, fast acid-producing lactococci were grouped as 61  Lactococcus lactis subsp. lactis , 13  L. lactis subsp. cremoris and 14  L. lactis subsp. lactis biovar diacetylactis. Randomly amplified polymorphic DNA (RAPD) fingerprinting of fast acid-producing lactococci, using two primers, resulted in 21 different RAPD patterns for L. lactis subsp. lactis isolates, nine RAPD patterns for L. lactis subsp. cremoris isolates and three RAPD patterns for L. lactis subsp. lactis biovar diacetylactis isolates. Up to 19 different RAPD patterns were found for L. lactis isolates from cheeses made in a particular month.     

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.     

Some technological properties of Penicillium roqueforti strains isolated from a home-made blue cheese

Nine strains of Penicillium roqueforti isolated from a traditional Spanish blue cheese (Valdeón cheese) along with two commercial strains were investigated for their ability to grow at different concentrations of salt and at different temperatures as well as for their proteolytic and lipolytic activities. Low concentrations of salt (1-3%) were stimulating for all the strains, with 1% salt being the concentration with the highest stimulating effect in nearly all. The rate of growth at 10°C was 2-3 times lower than at 25°C, the optimum temperature for the species. None of the strains, including the commercial cultures, showed proteolytic activity on casein agar, while all of them were lipolytic on tributyrin agar.     

Characterization of the Enterobacteriaceae isolated from an artisanal Italian ewe's cheese (Pecorino Abruzzese)

AIMS: To evaluate some physiological characteristics of the Enterobacteriaceae isolated from Pecorino cheese. METHODS AND RESULTS: The production of organic acids, secondary volatile compounds, biogenic amines (BA) and the lipolytic and proteolytic activities of Citrobacter braakii, Enterobacter sakazakii, Escherichia coli, Kluyvera spp., Salmonella enterica ssp. arizonae and Serratia odorifera strains were determined in skim milk after 48 h of fermentation at 30 degrees C. The proteolytic activity observed only in Ser. odorifera and Kluyvera spp. was confirmed by the peptide profiles of the pH 4.6-insoluble fraction using RP-HPLC; however, the lipase activity was evidenced in all the isolates of E. coli, Kluyvera spp. and Salm. enterica ssp. arizonae. During fermentation, all the strains utilized citric acid and produced significant quantities of putrescine followed by histamine, spermine and spermidine as well as acetic and lactic acid. Moreover, the major volatile compounds produced were ethanol, 2,3-butanedione, 3-hydroxy-2-butanone, 2-heptanone and acetone. CONCLUSIONS: The Enterobacteriaceae of dairy origin possess many metabolic activities that could affect the sensory quality of the cheese in which they grow during ripening. SIGNIFICANCE AND IMPACT OF THE STUDY: The important physiological characteristics possessed by Enterobacteriaceae confirm the complexity of the microbiota of Pecorino Abruzzese cheese, which influences the typical sensory properties of this product.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2–3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10⁷ cfu/g.     

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.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2-3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10(7) cfu/g.     

Phenotypic and genotypic diversity of yeasts isolated from water-buffalo Mozzarella cheese

Water-buffalo Mozzarella (WBM) cheese is one of the several 'pasta filata' or stretched curd cheeses that originated in southern Italy, traditionally manufactured from raw milk employing natural whey starter cultures. Lactose- and galactose-fermenting yeasts isolated from WBM were studied to evaluate their role in the ripening of this cheese. The kinetic parameters of the growth of the yeasts as well as their principal metabolic end-products showed a great variability depending on the species. Moreover, the genetic polymorphism of the yeasts was studied for their differentiation at species level by means of the polymerase chain reaction (PCR) fingerprinting and mitochondrial DNA (mtDNA) restriction analysis. While the differentiation based on metabolic traits was not able to discriminate Kluyveromyces marxianus, Candida kefyr and C. sphaerica, the PCR analysis with primers M13 and RF2 resulted in a reliable and rapid method for differentiating at species level Saccharomyces cerevisiae, K. marxianus, K. lactis and their anamorphic species. Furthermore, mtDNA analysis proved to be more discriminating at strain level.     

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.     

Identification of enterococci isolated from cow's milk cheese: comparison of the classical methods and the API 20 STREP system (technical note)

A comparison of the results obtained using the classical methods with those of the API 20 Strep system was carried out in identifying 24 enterococci strains isolated from San Simón cow's milk cheese, a traditional Spanish variety. The results of both identification systems coincided exactly in 9 strains (37.5% of the strains studied). In one strain the results obtained using the classical methods did not coincide with those using the API 20 Strep method. 3 strains (12.5%) could not be identified using the API 20 Strep system. However, 11 strains (45%), that remained doubtful between both species E. faecalis and E. faecium on the basis of the classical methods, were identified using the API 20 Strep system. The API 20 Strep system does not include some biochemical tests of importance in identifying of foodborne enterococci and could not identify the atypical strains of Enterococcus. Moreover, this system is adapted to the identification of enterococci of clinical origin and their database does not include some species common in foods. However, it could have an application in combination with the classical methods in order to carry out a reasonably rapid and reliable identification of enterococci related to cheese.     

Genetic diversity amongFrankia isolated from Casuarina nodules

The nodulation ability of variousFrankia strains isolated from the nodules of Casuarina were tested on two Casuarina species (C. equisetifolia andC. glauca), and onHippophaë rhamnoides. We found that the isolates could be separated into two groups, some of them being unable to reinfect the Casuarina host-plant but infective onH. rhamnoides. Other isolates effectively nodulated the original Casuarina host-plant. The second purpose of this study was to examine the genetic diversity among the Casuarina-isolated strains using well-characterized symbiotic genes as hybridization probes. We found a relationship between nodulation characteristics and hybridization patterns.     

Genetic diversity of Acanthamoeba isolated from ocean sediments

Genetic diversity of 18 Acanthamoeba isolates from ocean sediments was evaluated by comparing mitochondrial (mt) DNA RFLP, 18S rDNA sequences and by examining their cytopathic effects on human corneal epithelial cells versus reference strains. All isolates belonged to morphologic group II. Total of 16 restriction phenotypes of mtDNA from 18 isolates demonstrated the genetic diversity of Acanthamoeba in ocean sediments. Phylogenetic analysis using 18s rDNA sequences revealed that the 18 isolates were distinct from morphological groups I and III. Fifteen isolates showed close relatedness with 17 clinical isolates and A. castellanii Castellani and formed a lineage equivalent to T4 genotype of Byers group. Two reference strains from ocean sediment, A. hatchetti BH-2 and A. griffini S-7 clustered unequivocally with these 15 isolates. Diversity among isolates was also evident from their cytopathic effects on human corneal cells. This is the first time describing Acanthamoeba diversity in ocean sediments in Korea.     

A survey of the enterococci isolated from an artisanal Italian goat's cheese (semicotto caprino)

Enterococci were isolated from semicotto caprino cheese, a traditional cheese produced in Southern Italy: they were a significant part of the microbial population of this cheese, confirming the importance of the presence of these micro-organisms during cheese-making and ripening. They were also identified and studied for their phenotypic and genotypic characteristics: Enterococcus faecalis and Ent. faecium were the most frequently isolated species, followed by Ent. durans, Ent. hirae and Ent. gallinarum. None of the isolates showed lipolytic activity, whereas they were characterized by a relevant proteolytic activity as well as an antagonistic activity towards Listeria innocua. One strain of Ent. gallinarum showed a low-level resistance to vancomycin, while six out of the 79 Ent. faecalis strains possessed beta-haemolysis reaction. The highest acidifying potential in skim milk was obtained by Ent. faecalis isolates. Thirty enterococcal strains representative of the different species at different ripening times were analysed by means of RAPD-PCR, and revealed species-specific profiles for all the considered species.     

Genetic diversity and spoilage potentials among Pseudomonas spp. isolated from fluid milk products and dairy processing plants

Degradation of milk components through various enzymatic activities associated with the contamination of dairy products by Pseudomonas spp. can reduce the shelf life of processed milk. Reliable methods for differentiating among Pseudomonas spp. strains are necessary to identify and eliminate specific sources of bacterial contamination from dairy processing systems. To that end, we assessed the genetic diversity and dairy product spoilage potentials among a total of 338 Pseudomonas spp. isolates from raw and pasteurized milk and from environmental samples collected from four dairy processing plants. The majority of isolates were identified as P. fluorescens and P. putida by API 20 NE. A total of 42 different ribotype patterns were identified among a subset of 81 isolates. The presence of many different ribotypes within this collection indicates high genetic diversity among the isolates and suggests multiple origins of contamination within the processing plant and in dairy products. The extracellular enzyme activity patterns among Pseudomonas isolates appeared to be associated with ribotypes. Isolates with the same ribotype frequently had the same extracellular protease, lecithinase, and lipase activities. For example, isolates grouped in ribotype 55-S-6 had the highest extracellular protease activity, while those in ribotypes 50-S-8 and 72-S-3 had the highest extracellular lipase activities. We conclude that ribotyping provides a reliable method for differentiating Pseudomonas strains with dairy food spoilage potential.     

The behaviour of Enterobacteriaceae in Manchego cheese made from raw ewes' milk treated with hydrogen peroxide, potassium nitrate or potassium nitrite

Growth of Enterobacteriaceae, coliforms and faecal coliforms in Manchego cheese during the first 24 h after manufacture was retarded by treatment of milk with 0.1 g 1^-1 H₂O₂ compared to growth in control cheese made from untreated milk. Moreover, the decrease in their numbers during cheese ripening was accelerated by the H₂O₂ treatment of milk. In contrast, KNO₃ or KNO₂ addition to milk enhanced the growth of these micro-organisms during cheese manufacture and favoured their survival during ripening.     

Potential of enterococci isolated from horses

Faecal samples of 122 horses (from farms in Slovakia) were examined to select enterococci to study their probiotic potential for their further use as additives. Each gram of faeces contained 1.0-5.0cfu (log 10) of enterococci. Of the 43 isolates, 25 (58.1%) were identified as Enterococcus faecium, 3 strains were (6.9%) Enterococcus mundtii and one strain was identified as E. faecalis. Fourteen isolates were not characterized further. A significant proportion of the isolates were resistant to kanamycin, vancomycin and gentamicin. Low urease activity of enterococci dominated. The values of lactic acid ranged from 0.98 to 1.91mmol/L. Porcine fibronectectin and bovine lactoferrin were bound weakly by tested enterococci, while bovine fibrinogen was bound more strongly. Enterococci from horses did not bind bovine apotransferrin. The isolates adhered with the same ability to human as well as to canine mucus. At least one enterocin gene was detected among 16 analyzed isolates. Ent B gene was detected in all strains tested (16, 100%), followed by the genes ent A, ent P and ent L50B. Three suitable candidates-the strains of E. faecium EF 412, EF 462 and EF 491 were selected for further detail studies and possibilities to be used as additives.     

Genetic diversity of Bradyrhizobium strains isolated from Arachis hypogaea

Rhizobia are used exclusively in agricultural systems for enhancing the ability of legumes to fix atmospheric nitrogen. Knowledge about the indigenous population is necessary for the selection and application of inoculant strains. In this study, we have assessed the genetic diversity of Bradyrhizobium strains isolated from the host plant, Arachis hypogaea along the coastline of Tamil Nadu. Different populations collected from varying environmental conditions were analysed for salt and pH tolerance. Genetic diversity among the strains was studied using RAPD markers and PCR-RFLP of 16S rDNA and nifD genes. The approaches used in this study yielded consistent results, which revealed a high degree of heterogeneity among strains and detection of two distinct genetic groups.     

Genetic diversity in Selenmonas ruminantium isolated from the rumen

Abstract Non-denaturing polyacrylamide gel electrophoresis was used to detect genetic variation at loci coding for there intracellular enzymes in the obligately anaerobic rumen bacterium Selenomonas ruminantium . Four mobility variants were detected for lactate dehydrigenase, seven for glucokinase and at least five for NADP-dependent glutamate dehydrogenase among 28 newly isolated, and eitght previously isolated strains from sheep and cattle. No evidence was found for an exclusive association of any particular electrophoretic mobility type with variable metabolic traits such as the ability to utilise lactate, to reduce nitrate or to ferment trehalose, sorbitol, rhamnose or glycerol. The most commonly occurring electromorph type was recovered from more than one animal, while most animals examined were show to harbour more than one electromorph type.