Resistance against Industrial Bacteriophages Conferred on Lactococci by Plasmid pAJ1106 and Related Plasmids

Plasmid pAJ1106 and its deletion derivative, plasmid pAJ2074, conferred lactose-fermenting ability (Lac) and bacteriophage resistance (Hsp) at 30°C to Lac⁻ proteinase (Prt)-negative Lactococcus lactis subsp. lactis and L. lactis subsp. lactis var. diacetylactis recipient strains. An additional plasmid, pAJ331, isolated from the original source strain of pAJ1106, retained Hsp and conjugative ability without Lac. pAJ331 was conjugally transferred to two L. lactis subsp. lactis and one L. lactis subsp. cremoris starter strains. The transconjugants from such crosses acquired resistance to the phages which propagated on the parent recipient strains. Of 10 transconjugant strains carrying pAJ1106 or one of the related plasmids, 8 remained insensitive to phages through five activity test cycles in which cultures were exposed to a large number of industrial phages at incubation temperatures used in lactic casein manufacture. Three of ten strains remained phage insensitive through five cycles of a cheesemaking activity test in which cultures were exposed to approximately 80 different phages through cheesemaking temperatures. Three phages which propagated on transconjugant strains during cheesemaking activity tests were studied in detail. Two were similar (prolate) in morphology and by DNA homology to phages which were shown to be sensitive to the plasmid-encoded phage resistance mechanism. The third phage was a long-tailed, small isometric phage of a type rarely found in New Zealand cheese wheys. The phage resistance mechanism was partially inactivated in most strains at 37°C.     

Identification of Four Phage Resistance Plasmids from Lactococcus lactis subsp. cremoris HO2

The bacteriophage-host sensitivity patterns of 16 strains of Lactococcus lactis originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates, Lactococcus lactis subsp. cremoris HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage 712 (936 phage species) and the prolate-headed phage c2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system.     

Design of a Phage-Insensitive Lactococcal Dairy Starter via Sequential Transfer of Naturally Occurring Conjugative Plasmids

The plasmid-free Lactococcus lactis subsp. cremoris MG1614 is highly phage sensitive and lacks lactose fermenting ability (Lac) and primary casein degrading ability (Prt). Food grade gene transfer systems were used to sequentially superimpose different phage defense systems on this background, resulting in a gradual increase in resistance to bacteriophage in the derivatives. pLP712, encoding Lac and Prt, was then transferred to one of these hosts, into which plasmids encoding adsorption inhibition, restriction modification, and abortive infection had already been introduced. This resulted in a phage-resistant strain which was successfully used as a single-strain starter for cheddar cheese manufacture under industrial conditions.     

Detection and Viability of Lactococcus lactis throughout Cheese Ripening

Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 10⁶ to 10⁸ CFU of L. lactis per gram of product, thirteen from 10³ to 10⁵ CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 10⁵ to 10⁹ CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese.     

Interactions of Lactobacillus bulgaricus Temperate Bacteriophage 0448 with Host Strains

Lactobacillus bulgaricus LT4(0448) is a lysogenic strain from which a temperate bacteriophage can be induced by mitomycin C or UV irradiation. Lactobacillus lactis CNRZ 326 is an indicator strain for the temperate phage 0448, but this strain lyses only in the presence of Ca²⁺ ions. A resistant culture developed secondarily after phage lysis and grew normally in MRS broth but again lysed abruptly if Ca²⁺ ions were added after two or three transfers. This behavior of the secondary culture and its subcultures is explained by a heterogeneous and fluctuating bacterial population, including clones identical to L. lactis 326, which were sensitive to 0448 and which formed rough colonies, as does the indicator. The proportion of these clones increased in the course of transfers in MRS, explaining lysis when Ca²⁺ was added. The population also included clones which formed smooth colonies (S clones). SI clones, which could not be induced by mitomycin C, were the major type in the initial culture, although they were sensitive to temperate phage 0448. The SI population then decreased and was gradually replaced by SII clones, inducible by mitomycin C and resistant to 0448. These SII clones were lysogenized clones, 326(0448), whose stability was confirmed by growth in the presence of an antiphage serum. When L. bulgaricus LT4(0448) was treated with mitomycin C, several cured LT4 clones were obtained that were related to the clones of the indicator L. lactis 326; they formed rough colonies. They also became sensitive to lytic phages or temperate phages active against L. lactis 326 and insensitive to lytic phages which lysed L. bulgaricus LT4(0448). This suggests that phage 0448 can lead to a lysogenic conversion of host strain LT4.     

Conjugal Transfer of Bacteriophage Resistance Determinants on pTR2030 into Streptococcus cremoris Strains

Agar surface conjugal matings were used to introduce heat-sensitive phage resistance (Hsp⁺) determinants carried on the conjugal plasmid pTR2030 into Streptococcus cremoris KH, HP, 924, and TDM1. Lactose-fermenting (Lac⁺) transconjugants were selected from matings of Lac⁻ variants of S. cremoris KH, HP, 924, and TDM1 with Streptococcus lactis ME2 or a high-frequency donor, S. lactis T-EK1 (pTR1040, Lac⁺; pTR2030, Hsp⁺). For all of the S. cremoris strains examined, select Lac⁺ transconjugants were completely resistant to plaquing by their homologous lytic phages. In all cases the plaquing efficiencies were less than 10⁻⁹. Acquisition of a 30-megadalton plasmid (pTR2030) in the S. cremoris phage-resistant transconjugants was demonstrated by direct plasmid analysis, by hybridization with ³²P-labeled probes, or by conjugal transfer of pTR2030 out of the phage-resistant transconjugants into a plasmid-cured recipient, S. lactis LM2302. Acid production, coagulation ability, and proteolytic activity of phage-resistant transconjugants in milk were comparable to those of their phage-sensitive parents. Further, S. cremoris phage-resistant transconjugants were not attacked by phage in starter culture activity tests, which included a 40°C incubation period. The results demonstrated that phage resistance determinants on pTR2030 could be conjugally transferred to a variety of S. cremoris strains and confer resistance to phage under conditions encountered during cheese manufacture. Phage-resistant transconjugants of S. cremoris M43 and HP were also constructed without the use of antiblotic markers to select conjugal recipients from mating mixtures.     

Restriction and Modification in Group N Streptococci: Effect of Heat on Development of Modified Lytic Bacteriophage

The appearance of lytic bacteriophage against newly introduced starter strains used during commercial cheese manufacture occurs rapidly, and their origin is not well understood. In this study, members of the group N streptococci were examined for the presence of bacteriophage restriction and modification systems. Two streptococcal phages from Streptococcus cremoris TR and Streptococcus lactis C2 (phage designations tr and c2) showed restricted lytic development on S. cremoris 799 and KH, respectively. Efficiency of plaquing was 1.9 × 10⁻⁷ for tr plaqued on 799 and 2.1 × 10⁻⁷ for c2 plaqued on KH. After passage through the restrictive hosts, these phages demonstrated high lytic ability for formerly restrictive hosts. Stress of the restrictive host strains at temperatures of 40 to 50°C resulted in a significant increase in the efficiency of plaquing of restricted bacteriophages. Elevated temperatures are encountered during commercial cheese manufacture. The results suggested that the temporary loss of host restriction activity with the resulting modification of nonspecific bacteriophage may contribute directly to the appearance of lytic phage against new starter strains.     

Argentinean Lactococcus lactis bacteriophages: genetic characterization and adsorption studies

Aims: Characterization of four virulent Lactococcus lactis phages (CHD, QF9, QF12 and QP4) isolated from whey samples obtained from Argentinean cheese plants. Methods and Results: Phages were characterized by means of electron microscopy, host range and DNA studies. The influence of Ca²⁺, physiological cell state, pH and temperature on cell adsorption was also investigated. The double‐stranded DNA genomes of these lactococcal phages showed distinctive restriction patterns. Using a multiplex PCR, phage QP4 was classified as a member of the P335 polythetic species while the three others belong to the 936 group. Ca²⁺ was not needed for phage adsorption but indispensable to complete cell lysis by phage QF9. The lactococci phages adsorbed normally between pH 5 and pH 8, and from 0°C to 40°C, with the exception of phage QF12 which had an adsorption rate significantly lower at pH 8 and 0°C. Conclusions: Lactococcal phages from Argentina belong to the same predominant groups of phages found in other countries and they have the same general characteristics. Significance and Impact of the Study: This work is the first study to characterize Argentinean L. lactis bacteriophages.     

Inhibition of Listeria innocua in Cheddar Cheese by Addition of Nisin Z in Liposomes or by In Situ Production in Mixed Culture

The effect of addition of purified nisin Z in liposomes to cheese milk and of in situ production of nisin Z by Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in the mixed starter on the inhibition of Listeria innocua in cheddar cheese was evaluated during 6 months of ripening. A cheese mixed starter culture containing Lactococcus lactis subsp. lactis biovar diacetylactis UL719 was selected for high-level nisin Z and acid production. Experimental cheddar cheeses were produced on a pilot scale, using the selected starter culture, from milk with added L. innocua (10⁵ to 10⁶ CFU/ml). Liposomes with purified nisin Z were prepared from proliposome H and added to cheese milk prior to renneting to give a final concentration of 300 IU/g of cheese. The nisin Z-producing strain and nisin Z-containing liposomes did not significantly affect cheese production and gross chemical composition of the cheeses. Immediately after cheese production, 3- and 1.5-log-unit reductions in viable counts of L. innocua were obtained in cheeses with encapsulated nisin and the nisinogenic starter, respectively. After 6 months, cheeses made with encapsulated nisin contained less than 10 CFU of L. innocua per g and 90% of the initial nisin activity, compared with 10⁴ CFU/g and only 12% of initial activity in cheeses made with the nisinogenic starter. This study showed that encapsulation of nisin Z in liposomes can provide a powerful tool to improve nisin stability and inhibitory action in the cheese matrix while protecting the cheese starter from the detrimental action of nisin during cheese production.     

Transfer of Sucrose-Fermenting Ability and Nisin Production Phenotype among Lactic Streptococci

Transfer of sucrose fermentation ability, nisin production, and nisin resistance from Streptococcus lactis to S. lactis and Streptococcus lactis subsp. diacetylactis occurred between cells immobilized on nitrocellulose filters in the presence of DNase. Transconjugants were able to act as donors to transfer the Suc-Nis phenotype in subsequent mating. No changes in sensitivity to lytic phage c2 were noted in S. lactis transconjugants. However, temperature-independent restriction of lytic phage 18-16 was noted in transconjugants of S. lactis subsp. diacetylactis 18-16. Adsorption studies with phage-resistant transconjugants showed that resistance was not due to lack of adsorption by the lytic phage. Physical evidence for the presence of introduced plasmid DNA was not found in lysates of transconjugants.     

Multifactorial diversity sustains microbial community stability

Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty.     

Conjugal Transfer in Lactic Streptococci of Plasmid-Encoded Insensitivity to Prolate- and Small Isometric-Headed Bacteriophages

Eight of 40 strains of Streptococcus lactis and S. lactis subsp. diacetylactis were able to conjugally transfer a degree of phage insensitivity to Streptococcus lactis LM0230. Transconjugants from one donor strain, S. lactis subsp. diacetylactis 4942, contained a 106-kilobase (kb) cointegrate plasmid, pAJ1106. The plasmid was conjugative (Tra⁺) and conferred phage insensitivity (Hsp) and lactose-fermenting ability (Lac) in S. lactis and Streptococcus cremoris transconjugants. The phage resistance mechanism was effective against prolate- and small isometric-headed phages at 30°C. In S. lactis transconjugants, the phage resistance mechanism was considerably weakened at elevated temperatures. A series of deletion plasmids was isolated from transconjugants in S. cremoris 4854. Deletion plasmids were pAJ2074 (74 kb), Lac⁺, Hsp⁺, Tra⁺; pAJ3060 (60 kb), Lac⁺, Hsp⁺; and pAJ4013 (13 kb), Lac⁺. These plasmids should facilitate mapping Hsp and tra genes, with the aim of constructing phage-insensitive strains useful to the dairy industry.     

Genetic Control of Capsid Length in Bacteriophage T4 I. Isolation and Preliminary Description of Four New Mutants

Four new mutants are described whose phenotypic expression affects the length of the head of bacteriophage T4D. All mutants produce some phenotypically normal phage particles. Mutant pt21-34 also produces at least two size classes of phage particle which have heads that are shorter than normal. The other three mutants, ptg19-2, ptg19-80, and ptg191, produce, in addition to phages with normal and with shorter-than-normal heads, giant phages with heads from 1.5 to at least 10 times the normal length. All mutations are clustered near gene 23. Giant phage particles have the following properties: they are infectious and contain and inject multiple genomes as a single continuous bihelical DNA molecule of greater-than-unit length. Their frequency, relative to the total plaque-former population, increases late in the infectious cycle. They have a normal diameter, variable length, and a buoyant density range in CsCl from equal to slightly greater than that of normal phage. The arrangement of capsomers is visible in the capsids, which are composed of cleaved gene 23 protein.     

Cooperation between Lactococcus lactis and Nonstarter Lactobacilli in the Formation of Cheese Aroma from Amino Acids

In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of Lactococcus lactis subsp. cremoris NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of α-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than L. lactis subsp. cremoris NCDO763, and produced mainly nonaromatic metabolites. L. lactis subsp. cremoris NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced α-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with L. lactis subsp. cremoris NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the Lactococcus strain. Therefore, we think that such cooperation between starter L. lactis and GDH-positive lactobacilli can stimulate flavor development in cheese.     

Ultrastructure and Host Specificity of Bacteriophages of Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, and Leuconostoc cremoris from Finnish Fermented Milk “Viili”

“Viili,” a fermented milk product, has a firm but viscous consistency. It is produced with traditional mesophilic mixed-strain starters, which have various stabilities in dairy practice. Thirteen morphologically different types of phages were found in 90 viili samples studied by electron microscopy. Ten of the phage types had isometric heads with long, noncontractile tails, two had elongated heads with long, noncontractile tails, and one had a unique, very long elongated head with a short tail. Further morphological differences were found in the tail size and in the presence or absence of a collar, a baseplate, and a tail fiber. To find hosts for the industrially significant phages, we examined the sensitivities of 500 bacterial isolates from starters of the viili. Seven of the phages attacked Streptococcus cremoris strains, three attacked S. lactis subsp. diacetylactis strains, and four attacked Leuconostoc cremoris strains. Some phages differed only in their host specificity. Hosts were not found for 4 of the 13 morphological types of phages.     

Identification and Characterization of Lactococcal-Prophage-Carried Superinfection Exclusion Genes

Superinfection exclusion (Sie) proteins are prophage-encoded phage resistance systems. In this study, genes encoding Sie systems were identified on the genomes of Lactococcus lactis subsp. cremoris MG1363 and L. lactis subsp. lactis IL1403. These Sie systems are genetically distinct and yet were shown to act specifically against a particular subset of the 936 phage group. Each of the systems allows normal phage adsorption while affecting plasmid transduction and intracellular phage DNA replication, which points to the blocking of phage DNA injection as their common mode of action. Sie-specifying genes found on the MG1363 prophages are also present in various lactococcal strains, whereas the prophage-encoded Sie systems of IL1403 do not appear to be as widely disseminated.     

Use of a Genetically Enhanced, Pediocin-Producing Starter Culture, Lactococcus lactis subsp. lactis MM217, To Control Listeria monocytogenes in Cheddar Cheese

Cheddar cheese was prepared with Lactococcus lactis subsp. lactis MM217, a starter culture which contains pMC117 coding for pediocin PA-1. About 75 liters of pasteurized milk (containing ca. 3.6% fat) was inoculated with strain MM217 (ca. 10⁶ CFU per ml) and a mixture of three Listeria monocytogenes strains (ca. 10³ CFU per ml). The viability of the pathogen and the activity of pediocin in the cheese were monitored at appropriate intervals throughout the manufacturing process and during ripening at 8°C for 6 months. In control cheese made with the isogenic, non-pediocin-producing starter culture L. lactis subsp. lactis MM210, the counts of the pathogen increased to about 10⁷ CFU per g after 2 weeks of ripening and then gradually decreased to about 10³ CFU per g after 6 months. In the experimental cheese made with strain MM217, the counts of L. monocytogenes decreased to 10² CFU per g within 1 week of ripening and then decreased to about 10 CFU per g within 3 months. The average titer of pediocin in the experimental cheese decreased from approximately 64,000 arbitrary units (AU) per g after 1 day to 2,000 AU per g after 6 months. No pediocin activity (<200 AU per g) was detected in the control cheese. Also, the presence of pMC117 in strain MM217 did not alter the cheese-making quality of the starter culture, as the rates of acid production, the pH values, and the levels of moisture, NaCl, and fat of the control cheese and the experimental cheese were similar. Our data revealed that pediocin-producing starter cultures have significant potential for protecting natural cheese against L. monocytogenes.     

Genetic Variation of Lactobacillus delbrueckii subsp. lactis Bacteriophages Isolated from Cheese Processing Plants in Finland

The genomes of four Lactobacillus delbrueckii subsp. lactis bacteriophages were characterized by restriction endonuclease mapping, Southern hybridization, and heteroduplex analysis. The phages were isolated from different cheese processing plants in Finland between 1950 and 1972. All four phages had a small isometric head and a long noncontractile tail. Two different types of genome (double-stranded DNA) organization existed among the different phages, the pac type and the cos type, corresponding to alternative types of phage DNA packaging. Three phages belonged to the pac type, and a fourth was a cos-type phage. The pac-type phages were genetically closely related. In the genomes of the pac-type phages, three putative insertion/deletions (0.7 to 0.8 kb, 1.0 kb, and 1.5 kb) and one other region (0.9 kb) containing clustered base substitutions were discovered and localized. At the phenotype level, three main differences were observed among the pac-type phages. These concerned two minor structural proteins and the efficiency of phage DNA packaging. The genomes of the pac-type phages showed only weak homology with that of the cos-type phage. Phage-related DNA, probably a defective prophage, was located in the chromosome of the host strain sensitive to the cos-type phage. This DNA exhibited homology under stringent conditions to the pac-type phages.     

Protein characterization of lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages isolated from cheese wheys

Proteins of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages were studied using antibody inhibition assay and immunoblotting. Antisera were prepared against four representative L. lactis ssp. lactis and L. lactis ssp. cremoris phages (D59-1, F4-1, G72-1, and I37-1), which were selected from 17 isolates, derived from commercial cheese wheys. The reactivities of the four antisera with 13 other phage isolates were tested. Among these isolates, two phage groups having distinct serological properties were found. Group I reacted with the antisera against phages D59-1/F4-1 and Group II reacted with the antisera against phages G72-1/I37-1. Strongly lytic phages, capable of lysing phage-resistant host strains, were found to share protein similarities with the phage protein group I, and phages isolated from phage-sensitive host strains belonged to the phage protein group II. Furthermore, group I was composed of all prolate and some isometric phages, whereas group II was composed solely of the isometric phages. Thus, the two serologically distinct phage groups were not correlated with the two morphological groups, prolate and isometric. Proteins of the four phages were further characterized by immunoblotting and silver staining. A 22.5-kDa antigenic polypeptide of phage I37-1, and three polypeptides of 65, 37, 21 kDa in phage F4-1 were responsible for the cross-reactivities in group II and group I, respectively. Correspondence to: R. A. Ledford     

An Ecological Study of Lactococci Isolated from Raw Milk in the Camembert Cheese Registered Designation of Origin Area

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of “Camembert de Normandie” cheese.