Antimicrobial activity of enterococci strains isolated from white cheese

Twelve strains of enterococci isolated from white cheese samples obtained from different regions of Turkey were screened for their antimicrobial activity against some Gram-positive and Gram-negative food-borne pathogens and contaminating bacteria, also against themselves and some other lactic acid bacteria (LAB), including some species of lactococci, lactobacilli and Leuconostoc . Antimicrobial activity was confirmed for all 12 strains, including six Enterococcus faecium , two Enterococcus faecalis and four Enterococcus durans . Antimicrobial agents produced by these enterococci exhibited a spectrum of activity, which is mainly directed against food-borne pathogens, specially Listeria monocytogenes and Bacillus cereus and some other Bacillus spp. and also some enterococci. Tested LAB was insensitive to inhibitory agents produced by them.     

Phenotypic,genetic and technological characterization of Lactococcus garvieae strains isolated from a raw milk cheese

A series of Lactococcus garvieae strains isolated as the majority population of a Spanish traditional, starter-free cheese made from raw milk were phenotypically and genotypically characterised to address their biochemical potential, safety requirements, and technological properties. As expected, all L. garvieae cheese strains fermented lactose but grew slowly in UHT-treated milk. Enzymatic activities of L. garvieae were similar to those of Lactococcus lactis, although higher esterase and lipase activities were recorded for L. garvieae strains. Profiles of the volatile compounds produced from milk by L. garvieae and L. lactis strains were also comparable. L. garvieae strains did not produce haemolysin, gelatinase and the biogenic amines tyramine and histamine. Five L. garvieae stains showed tetracycline resistance encoded by a tet(M) gene. The use of L. garvieae strains as starter or adjunct cultures might be recommended for certain cheese types, provided that the safety of the strains has been demonstrated.     

Genotypic and technological characterization of enterococci isolated from artisanal Fiore Sardo cheese

A total of 118 enterococcal strains isolated from artisanal Fiore Sardo cheese were characterized technologically and genetically. The presence of potential virulence factors was also investigated. Strains were classified as Ec. faecium (84 strains), Ec. durans (24 strains) and Ec. faecalis (10 strains). RAPD-PCR analysis with two different primers (M13 and XD9) confirmed species identification and proved useful for the detection of interstrain variations, especially among Ec. faecium isolates. Most strains could hydrolyse casein and had weak acidifying activity in milk. None of the isolates produced lipolytic reactions. Gelatinase activity was observed in two strains of Ec. faecalis and one strain of Ec. durans. beta-Haemolysis on horses' blood was never detected in any of the strains, independently of species. Most strains produced tyramine from tyrosine but none decarboxylated lysine, histidine or ornithine. Overall, a wide spectrum of resistance was observed. Almost all strains were resistant to the aminoglycosides, gentamycin, kanamycin, streptomycin, neomycin, and to the semisynthetic penicillin, oxacillin, but resistance to vancomycin was not widespread among our strains: only one Ec. faecium and one Ec. durans strain were found to be vancomycin resistant. Our results show a certain diversity in technological traits of the enterococcal strains isolated from artisanal Fiore Sardo, together with a low incidence of some potentially pathogenic traits of health concern.     

Identification of tet(M) in two Lactococcus lactis strains isolated from a Spanish traditional starter-free cheese made of raw milk and conjugative transfer of tetracycline resistance to lactococci and enterococci

Specific PCR and sequencing showed that a tet(M) gene was present in two tetracycline-resistant Lactococcus lactis strains isolated from a raw milk, starter-free cheese. Hybridisation experiments using as a probe an internal segment of the gene obtained by PCR associated tet(M) with plasmids of around the same size (30 kbp) in both strains. Molecular analysis of the tetracycline resistance loci, including the upstream and downstream regions of the genes, showed them to be identical to one other and to the tet(M) encoded by the conjugative transposon Tn916. Amplification of Tn916-derived segments suggested the transposon was complete in the two L. lactis strains. Further, curing of the tetracycline resistance was accompanied by a reduction in size of the plasmids comparable to that expected for Tn916. Tetracycline resistance could be transferred by conjugation to plasmid-free Lactococcus and Enterococcus strains. However, no plasmid DNA was detected among the transconjugants while both tet(M) and transposon-related sequences were amplified by PCR. This suggested that only the transposon was mobilized.     

Virulence factors and genetic variability of Staphylococcus aureus strains isolated from raw sheep's milk cheese

Contamination of dairy products with Staphylococcus aureus can be of animal or human origin. The host pathogen relationship is an important factor determining genetic polymorphism of the strains and their potential virulence. The aim of the present study was to carry out an extensive characterization of virulence factors and to study the genetic variability of S. aureus strains isolated from raw ewe's milk cheese. A total of 100 S. aureus strains isolated from cheese samples produced in 10 artisan cheese factories were analyzed for the presence of enterotoxins (sea-see) and enterotoxins-like genes (seh, sek, sel, sem, seo, sep), leukocidins, exfoliatins, haemolysins, toxic shock syndrome toxin 1 (TSST-1) and the accessory gene regulator alleles (agr). Strains were also typed using pulsed-field gel electrophoresis (PFGE). AMOVA analysis carried out on PFGE and PCR data showed that the major component explaining genetic distance between strains was the dairy of origin. Of the total isolates 81% had a pathogenicity profile ascribable to "animal" biovar while 16% could be related to "human" biovar. The biovar allowed to estimate the most likely origin of the contamination. Minimum inhibitory concentrations (MICs) of nine antimicrobial agents and the presence of the corresponding genes coding for antibiotic resistance was also investigated. 18 strains carrying blaZ gene showed resistance to ampicillin and penicillin and 6 strains carrying tetM gene were resistant to tetracycline. The presence of mecA gene and methicillin resistance, typical of strains of human origin, was never detected. The results obtained in the present study confirm that S. aureus contamination in artisan cheese production is mainly of animal origin.     

Technological characterization of Geotrichum candidum strains isolated from a traditional Spanish goats' milk cheese

Forty-one strains of Geotrichum candidum isolated from Armada cheese, Sobado variety, were screened for their enzymatic activities, including proteolytic and lipolytic activities and aminopeptidase activity. The highest extracellular proteolytic activity was detected for 8 strains with values ranging between 2.086 and 4.685 mM Gly L⁻¹ of milk. Extracellular lipolytic activity was high for all but one of the G. candidum strains, with values ranging between 67 and 131 μmol oleic mL⁻¹. Cell-bound lipase activity showed values which were considerably lower than those for extracellular activity, ranging between 32.50 and 42.50 μmol oleic mL⁻¹ and falling below 20 μmol oleic mL⁻¹ in 28 strains. Aminopeptidase activity was not very high in the cell-free extract (CFE) of any of the strains, showing the highest values toward the substrate Lys-p-nitroanilide, with levels of activity ranging between 6.12 and 10.05 UE mg⁻¹ protein in 8 strains. In accordance with the results obtained, four G. candidum strains were selected as co-cultures in order to study their role in the ripening of a semi-hard goat’s milk cheese.     

Characterization of Staphylococcus aureus strains isolated from raw milk utilized in small-scale artisan cheese production

Staphylococcus aureus is an important agent of bacterial mastitis in milking animals and of foodborne intoxication in humans. The purpose of this study was to examine the genetic and phenotypic diversity, enterotoxigenicity, and antimicrobial resistance of S. aureus strains isolated from raw milk used for the production of artisan cheese in Vermont. Cross-tabulations revealed that the 16 ribotypes identified among the 90 milk isolates examined were typically associated with a specific animal species and that more than half of these ribotypes were unique to individual farms. In general, specific EcoRI ribotypes were commonly associated with specific phenotypical characteristics, including staphylococcal enterotoxin production or the lack thereof. Limited antimicrobial resistance was observed among the isolates, with resistance to ampicillin (12.51%) or penicillin (17.04%) most common. Two isolates of the same ribotype obtained from the same farm were resistant to oxacillin with 2% NaCl. More than half (52.22%) of isolates produced toxin, and 31 of the 32 isolates solely produced staphylococcal enterotoxin type C. Although these data demonstrate that S. aureus strains found in raw milk intended for artisan cheese manufacture are capable of enterotoxin production, staphylococcal enterotoxin C is not typically linked to foodborne illness. Because S. aureus is a common contaminant of cheese, an understanding of the ecology of this pathogen and of the antimicrobial susceptibility and toxigenicity of various strains will ultimately contribute to the development of control practices needed to enhance the safety of artisan and farmstead cheese production.     

Biodiversity and characterization of indigenous coagulase-negative staphylococci isolated from raw milk and cheese of North Italy

The aim of the current study was to detect coagulase-negative staphylococci (CNS) in raw milk and cheeses produced in North Italy, and to analyze isolates for their biodiversity, safety aspects and technological properties. Molecular identification methods revealed a high biodiversity among isolates and assigned them to 17 species. The most recovered species were Staphylococcus equorum (12%), Staphylococcus lentus (12%), Staphylococcus simulans (12%), Staphylococcus sciuri (10%), and Staphylococcus xylosus (9%). The presence of ten transferable antibiotic resistance (AR) genes was verified by PCR and 19% of isolates were positive, with tet(K) being the most frequent gene (10%); interestingly, no strain carried multiple AR genes. Twenty-four isolates displayed hemolytic activity; tyrosine decarboxylase gene (tdcA) was found in two isolates, while histidine decarboxylase gene (hdcA) and enterotoxin genes (se) were not detected. Isolates were further characterized for the presence of some relevant technological properties; 16% of isolates displayed proteolytic activity and 39% lipolytic activity, while no one of the isolates was found to exhibit antimicrobial activity against Staphylococcus aureus. This study provided evidence of a low occurrence of safety hazards in CNS isolated from dairy products.     

干酪中肠球菌种群的遗传差异及万古霉素抗性基因分析

通过种、属引物特异性扩增、重复序列PCR（rep-PCR）技术和万古霉素抗性基因检测对新疆北疆地区干酪样品中球菌的遗传结构差异进行分析。结果表明,15份样品共分离52株肠球菌,包括31株耐久肠球菌(Enterococcus durans)、18株粪肠球菌(Enterococcus faecalis)和3株屎肠球菌(Enterococcus faecium)。依据rep-PCR遗传指纹带谱分析,52株菌可以聚类成7个群,其中4个由E. durans构成。24株肠球菌检测到了万古霉素抗性基因,17株E. durans为VanC2/C3型,4株E. faecalis为VanC1型,2株E. faecium为VanB型,只有1株E. faecium为VanA型。新疆北疆地区干酪中肠球菌种群分布较为广泛,地域之间优势种群和基因型不同,同种肠球菌菌株之间存在广泛的遗传差异。     

Evaluation of metabolic activities of enterococci isolated from Pecorino Abruzzese cheese

Enterococci, which represent a considerable part of the natural microbiota of Pecorino Abruzzese, were investigated for biochemical activities considered to have a potential role in cheese ripening. Acidifying activity was weak, while interesting differences were identified for proteolytic and peptidolytic capability, particularly on H-Lys-β-NA. The presence of C-4 esterase and C-8 esterase lipase, as well as enzymes such as phosphohydrolases and acid phosphatase was confirmed by means of API ZYM. SPME-GC analyses of volatile compounds revealed the production of ethanol, diacetyl and, of particular interest, acetoin after 15 days at 10 °C, with important differences among the species. After 15 days at 10 °C, Enterococcus faecalis isolates were generally the most active, giving the highest results for most of the biochemical activities investigated, and also some Enterococcus durans showed interesting metabolic features that might be important to the development of the peculiar sensory properties of this cheese.     

Phenotypic and genetic characterization of a selected set of Lactococcus lactis strains isolated from a starter-free farmhouse cheese

Eleven strains of lactococci isolated from a farmhouse starter-free cheese manufactured from raw cow's milk were analysed in detail for some technologically-related properties. Large phenotypic differences were encountered between the isolates, some of which could be of practical relevance. Several strains produced lactic acid at a rate and at a final concentration suitable for large-scale cheesemaking. The enzymatic capabilities assayed with the API ZYM system showed that all strains possess similar profiles with weak proteinase and moderate leucine-arylamidase and esterase-lipase activities. Interestingly, two related strains presented a strong β -galactosidase activity. Plasmid and chromosomal analyses indicated a high degree of diversity among wild strains and showed low homology with some well-known Lactococcus lactis strains. Under highly stringent conditions, only one plasmid from a single strain gave a clear positive hybridization signal with lactococcal-derived probes for the β -phospho- galactosidase and the proteinase genes. In general, wild strains produced more odorous compounds and in higher amounts than the reference strains.     

Lactobacillus strains isolated from Danbo cheese as adjunct cultures in a cheese model system

Isolates of Non-Starter Lactic Acid Bacteria (NSLAB) from six ripened Danbo cheeses of different ages and of different brands were examined. Special emphasis was on the genus Lactobacillus with the aim of investigating their role in cheese maturation. Thirty-three isolates were typed by the PCR-based method, Randomly Amplified Polymorphic DNA (RAPD). Ten RAPD types were found and 70% of the isolates were of RAPD types found in more than one cheese. The different RAPD types were identified to species level by Temporal Temperature Gradient Gel Electrophoresis (TTGE). Most of the isolates were identified as Lactobacillus paracasei (76%), but also Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus rhamnosus and some taxa originating from the starter culture were detected. In one cheese, no lactobacilli were found.One strain of the most frequent Lactobacillus RAPD type from each of the five cheeses with a Lactobacillus flora was used as adjunct cultures in a cheese model system. Four of the five adjuncts were re-isolated during ripening. Two adjunct containing model cheeses received higher flavour scores than the control while two other were associated with off-flavours. The two model cheeses with off-flavour had a similar microflora and both were after 13 weeks of ripening dominated by a strain identified as L. plantarum.     

Short communication: Identification and technological characterization of yeast strains isolated from samples of water buffalo Mozzarella cheese

Sixty yeast cultures were isolated from samples of water buffalo Mozzarella, a popular “pasta filata” cheese, originating on 16 farms located in the provinces of Salerno, Caserta, and Frosinone (Italy). Strains were identified by means of 5.8S internal transcribed spacer rDNA PCR-RFLP combined with 26S rRNA gene partial sequencing and characterized for their ability to exert biochemical properties of technological interest. The recorded dominance of fermenting yeasts such as the lactose-fermenting Kluyveromyces marxianus (38.3% of the total isolates) and the galactose-fermenting Saccharomyces cerevisiae (21.6% of the total isolates) suggests that these yeasts contribute to the organoleptic definition of the water buffalo Mozzarella. The speciographic analysis revealed the presence of 7 other species rarely or never reported in a dairy environment belonging to the genera Pichia and Candida, whose role in Mozzarella cheese organoleptic properties need to be further investigated.     

Organic acids produced by lactobacilli, enterococci and yeasts isolated from Picante cheese

 Four species of bacteria (Enterococcus faecium and Enterococcus faecalis, Lactobacillus plantarum and Lactobacillus paracasei) and three species of yeasts (Debaryomyces hansenii, Yarrowia lipolytica and Cryptococcus laurentii), previously isolated from Picante cheese, were cultured in ovine and in caprine milk and assayed for sugar and organic acids metabolism for 6 days. The results indicated that both milk types can be coagulated by the four strains of lactic acid bacteria. Lb. paracasei led to a faster and greater reduction in pH. Production of lactic acid correlated to lactose degradation, and was highest for Lb. paracasei followed by E. faecium; citrate metabolism was apparent for E. faecalis and, to a lesser extent, for E. faecium, Lb. plantarum and Lb. paracasei. Relatively high contents of formic acid were found when inoculation was with Enterococcus and with Lb. plantarum. Received: 4 February 1999     

Identification and characterization of Saccharomyces cerevisiae strains isolated from West African sorghum beer

The occurrence and characterization of yeasts isolated from sorghum beer produced in Ghana and Burkina Faso, West Africa, were investigated. The yeasts involved in the fermentations were found to consist of Saccharomyces spp. almost exclusively. Of the isolates investigated, 45% were identified as Saccharomyces cerevisiae, whereas more than half of the isolates (53%) had physiological properties atypical of S. cerevisiae or any other member of the complex sensu strictu, as they were able to assimilate only glucose, maltose and ethanol as carbon sources. Both ITS-PCR RFLP and PFGE strongly indicated that these isolates were related to S. cerevisiae, regardless of their phenotypic characteristics. Sequencing of the D1/D2 domain of the 26S rDNA confirmed the close relatedness to S. cerevisiae with 0.5% nucleotide differences. The MAL1 and MAL3 loci were found for all isolates as the only recognized MAL loci. Besides, for 40% of the isolates the MAL61 probe hybridized to a position of about 950 kbp, which has not formerly been described as a MAL locus. The results showed that the spontaneous fermentation of West African sorghum beer is dominated by a variety of strains of S.cerevisiae not previously described, among which starter cultures should be selected.     

Identification of proteolytic pseudomonads isolated from raw milk

Proteolytic pseudomonads isolated from raw milk were classified by numerical taxonomy. Unweighted pair-group average-linkage cluster analysis was used to cluster 49 bacterial strains, of which 26 were Pseudomonas species, as described in the Bergey's Manual of Determinative Bacteriology, based on 52 characters. The milk isolates resided in two clusters; one containing Pseudomonas fluorescens and the other Pseudomonas fragi. The isolates identified with Pseudomonas fluorescens hydrolyzed milk proteins and milk fat and produced phospholipase. The Pseudomonas fragi-like isolates hydrolyzed milk proteins and milk fat but did not produce phospholipase and fluorescent pigment. The hydrolytic characteristics of milk isolates showed that the nature of the substrate and conditions under which the test was conducted were critical.     

Antibiotic resistance and virulence traits of enterococci isolated from Baylough, an Irish artisanal cheese

Eight representative Enterococcus strains from a collection of over 600 previously isolated from an Irish artisanal cheese were subjected to phenotypic and genotypic analysis of antibiotic resistance and virulence properties. Genes encoding resistance to tetracycline (tet(M) and tet(L)) and/or erythromycin (erm(B)) were detected in five strains. In addition, all strains contained two or more of the virulence genes tested (agg, gel, cyl, esp, ace, efaAfs, and efaAfm). Further investigation into the transferability and environmental dissemination of these resistance and virulence traits will allow risk assessment and safety evaluation of artisanal cheeses.     

Prevalence and characterization of Listeria monocytogenes isolated from soft cheese

A survey was made in 1995–1996 for Listeria spp. in 63 soft cheeses, made from raw ewe's milk using traditional methods, in the Province of Beira Baixa (Portugal). Listeria spp. were isolated from 47 (75%) of the cheeses, L.monocytogenes was isolated from 29 (46%), and L.innocua but not L.monocytogenes from 18 (29%). Of 24 isolates of L.monocytogenes that were serotyped, 20 were serotype 4b, three were serotype 1/2b and one was serotype 1/2a. Phage typing of isolates of L.monocytogenes and L.innocua showed that in some cases a particular phage type was associated with cheese from a particular source. Twenty four strains of L.monocytogenes tested were able to grow at 30°C in culture medium adjusted with HCl to a pH in the range from 4.4 to 6.0 within 3 days; in the pH range 4.4–6.8 a representative strain grew most rapidly at pH 6.8. The pH range in the cheeses during maturation was between about 5.2–6.4. Whether L.monocytogenes could multiply in the cheeses would depend on factors such as concentration of organic acids and of salt, and storage temperature.