Genetic diversity of the natural strains of <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis>

The method of RAPD-PCR and comparative analysis of the PCR fingerprinting profiles similarity was used to characterize interspecific diversity of natural isolates of the lactic acid bacteria Streptococcus thermophilus. The strain genetic diversity was demonstrated using three primer variants, designed for different bacterial genome regions. The resolution of RAPD-PCR technique with different primers for identification at the species level and for certification at the strain level, was examined relative to the commercially important cultures of S. thermophilus. The results provided conclusion on preferable usage of RAPD-PCR with the primer ERIC-1 for specific identification of S. thermophilus, and with the primer M13 for certification of natural isolates of this species at the strain level.     

Molecular characterization of lactic acid bacteria from sourdough breads produced in Sardinia (Italy) and multivariate statistical analyses of results

The objective of this work was to investigate the structure and diversity of lactic acid bacteria (LAB) communities in sourdough used for the production of traditional breads (Carasau, Moddizzosu, Spianata, Zichi) in Sardinia. 16S rDNA sequencing and Randomly Amplified Polymorphic DNA (RAPD-PCR) was applied for the identification and typing of the LAB isolated from 25 samples of sourdoughs. Multivariate statistical techniques were applied to RAPD-PCR pattern to study the biological diversity of sourdough samples. Twelve different species of LAB were identified, and most isolates were classified as facultative heterofermentative lactobacilli. Lactobacillus pentosus dominated the lactic microflora of many samples while Lactobacillus sanfranciscensis was isolated only from a limited number of samples. Although heterofermentative species represented between between 30% and 60% of the isolates in Carasau, Spianata and Zichi sourdoughs, only 2% of the isolates from Moddizzosu sourdoughs were identified as heterofermentative LAB. RAPD-PCR with a single primer followed by cluster analysis did not allow the identification of the isolates at the species level. However, a multidimensional scaling/bootstrapping approach on the RAPD-PCR patterns uncovered the diversity of the LAB communities of LAB showing differences both within and between bread types.     

Genetic diversity of the genus Lactobacillus bacteria from the human gastrointestinal microbiome

The species and strain genetic diversity of bacterial cultures belonging to the genus Lactobacillus, which were isolated from the gastrointestinal microbiome of the human population living in the former Soviet Union in the years 1960-1980, was studied. The bacteria demonstrated probiotic characteristics. Phylogenetic analysis of sequences of the gene coding for 16S rRNA detected earlier by us, showed that the gene found in bacteria isolated from the intestinal content of healthy adults and represented by species L. plantarum, L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum has high homology (97-100%) with this gene in type representatives of the species. The genotypic and strain diversity of cultures was studied using RAPD-PCR and nonspecific primers. This method with the use of the ERIC-1 primer gave reliable and reproducible results as compared that using with M13 and MSP primers and allowed the identification of examined bacteria belonging to the genus Lactobacillus at the level of species and certification at the strain level.     

Genetic diversity of the genus <Emphasis Type="Italic">Lactobacillus</Emphasis> bacteria from the human gastrointestinal microbiome

The species and strain genetic diversity of bacterial cultures belonging to the genus Lactobacillus, which were isolated from the gastrointestinal microbiome of the human population living in the former Soviet Union in the years 1960–1980, was studied. The bacteria demonstrated probiotic characteristics. Phylogenetic analysis of sequences of the gene coding for 16S rRNA detected earlier by us, showed that the gene found in bacteria isolated from the intestinal content of healthy adultsand represented by species L. plantarum, L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum has high homology (97–100%) with this gene in type representatives of the species. The genotypic and strain diversity of cultures was studied using RAPD-PCR and nonspecific primers. This method with the use of the ERIC-1 primer gave reliable and reproducible results as compared that using with M13 and MSP primers and allowed the identification of examined bacteria belonging to the genus Lactobacillus at the level of species and certification at the strain level.     

Comparison of statistical methods for identification of Streptococcus thermophilus, Enterococcus faecalis, and Enterococcus faecium from randomly amplified polymorphic DNA patterns

Thermophilic streptococci play an important role in the manufacture of many European cheeses, and a rapid and reliable method for their identification is needed. Randomly amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) with two different primers coupled to hierarchical cluster analysis has proven to be a powerful tool for the classification and typing of Streptococcus thermophilus, Enterococcus faecium, and Enterococcus faecalis (G. Moschetti, G. Blaiotta, M. Aponte, P. Catzeddu, F. Villani, P. Deiana, and S. Coppola, J. Appl. Microbiol. 85:25-36, 1998). In order to develop a fast and inexpensive method for the identification of thermophilic streptococci, RAPD-PCR patterns were generated with a single primer (XD9), and the results were analyzed using artificial neural networks (Multilayer Perceptron, Radial Basis Function network, and Bayesian network) and multivariate statistical techniques (cluster analysis, linear discriminant analysis, and classification trees). Cluster analysis allowed the identification of S. thermophilus but not of enterococci. A Bayesian network proved to be more effective than a Multilayer Perceptron or a Radial Basis Function network for the identification of S. thermophilus, E. faecium, and E. faecalis using simplified RAPD-PCR patterns (obtained by summing the bands in selected areas of the patterns). The Bayesian network also significantly outperformed two multivariate statistical techniques (linear discriminant analysis and classification trees) and proved to be less sensitive to the size of the training set and more robust in the response to patterns belonging to unknown species.     

Comparison of genotypic and biochemical characteristics of Streptococcus thermophilus strains isolated from sour milk products

Overall, 72 strains of lactic acid thermophilic streptococci isolated from sour milk products manufactured in various regions of Russia and European countries were analyzed using classical microbiological and molecular biological methods. Physiological and biochemical properties and genetic diversity of these Streptococcus thermophilus strains were studied, and a comparative analysis of the nucleotide sequences of the 16S rRNA gene was conducted. It has been demonstrated that the homology of proximal parts of the 16S rRNA gene of all the strains studied towards one another and towards the reference strain ATCC19258 amounts to 100%. As for the sugar fermentation, some strains display the characteristics untypical of the S. thermophilus members. The data obtained suggest that it is preferable to use gene 16S rRNA sequencing data for identification of natural isolates of closely related lactic acid bacterial species; moreover, this method is recommended for a precise species identification of industrial bacterial strains used in the food industry.     

Comparison of cultural methods for the identification and molecular investigation of yeasts from sourdoughs for Italian sweet baked products

Twenty-five yeast strains isolated from sourdough samples for Panettone, Pandoro and Cornetto brioche manufactured by eight different bakeries in northern Italy were characterised. Classification was performed by the simplified identification method (SIM), Kurtzman and Fell's identification protocol, the API system from bioMérieux (France) and the MicroLog system from Biolog (USA). Genetic diversity was investigated by randomly amplified polymorphic DNA fingerprinting and mitochondrial-DNA restriction enzyme analysis. Sequences of the internal transcribed spacers between 18S and 26S rDNA genes were analysed. Candida humilis was the predominant species (56% of isolates), whereas the remaining strains (44%) were related to the Saccharomyces cerevisiae sensu stricto group. Identification systems based on phenotypic analysis proved to be unreliable to identify yeasts from sourdough. Either RAPD-PCR or mtDNA restriction analysis showed to be suitable for the identification of species, but could not be used to differentiate among the isolates at the strain level. Sequencing of the ITS region permitted a consistent classification of the sourdough yeasts.     

Lactic acid bacteria diversity in corn silage produced in Minas Gerais (Brazil)

The diversity of lactic acid bacteria (LAB) in silages produced in warm climate countries is not well known. This study aimed to identify and characterise the metabolic and genotypic aspects of autochthonous LAB isolated from corn silage produced in the state of Minas Gerais, Brazil. Eighty-eight LAB were isolated. To evaluate their performance at the strain level, all isolates were distinguished among strains using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) and repetitive extragenic palindromic PCR (REP-PCR) techniques. The organic acid and ethanol production were determined by high-performance liquid chromatography (HPLC). The fingerprints obtained by RAPD-PCR with a M13 primer were more discriminatory than those obtained with the REP-PCR technique using a (GACA)4 primer. Moreover, 28 representative isolates were identified as Lactobacillus acidophilus, L. buchneri, L. casei, L. diolivorans, L. hilgardii, L. paracasei, L. parafarraginis, L. plantarum, L. rhamnosus, L. zeae and Pediococcus acidilactici. Different fingerprinting profiles between isolates within the same species were observed. However, some strains isolated from different silages showed the same band profile, thus suggesting the presence of clusters with high similar fingerprints in silages from various regions. A variation in LAB diversity was observed in the silages of the evaluated regions, with L. rhamnosus and L. buchneri showing the highest distribution. Differences in organic acid production were observed among the strains belonging to the same species. This research contributes to a better understanding of the LAB community present in corn silage produced in warm climates. These strains will be studied as potential silage starters.     

Comparison of a randomly amplified polymorphic DNA (RAPD) analysis and ATB ID 32C system for identification of clinical isolates of different Candida species

The objective of this work was to compare the usefulness of a randomly amplified polymorphic DNA (RAPD) assay to that of the ATB ID32C kit (bioMérieux, France) for identification of different species of Candida isolated from clinical specimens. The RAPD-PCR patterns obtained with OPE-18 primer for identification of clinical isolates were consistent, and the different independent assays revealed reproduction of the band patterns. RAPD with the OPE-18 primer is a very specific and sensitive method for identification of Candida glabrata, Candida guilliermondii, Candida tropicalis, Candida pelliculosa, Candida albicans, Candida krusei, and Candida lusitaniae.     

Rapid identification of dairy lactic acid bacteria by M13-generated, RAPD-PCR fingerprint databases

About a thousand lactic acid bacteria (LAB) isolated from dairy products, especially cheeses, were identified and typed by species-specific PCR and RAPD-PCR, respectively. RAPD-PCR profiles, which were obtained by using the M13 sequence as a primer, allowed us to implement a large database of different fingerprints, which were analysed by BioNumerics software. Cluster analysis of the combined RAPD-PCR fingerprinting profiles enabled us to implement a library, which is a collection of library units, which in turn is a selection of representative database entries. A library unit, in this case, can be considered to be a definable taxon. The strains belonged to 11 main RAPD-PCR fingerprinting library units identified as Lactobacillus casei/paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus brevis, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus and Lactococcus lactis. The possibility to routinely identify newly typed, bacterial isolates by consulting the library of the software was valued. The proposed method could be suggested to refine previous strain identifications, eliminate redundancy and dispose of a technologically useful LAB strain collection. The same approach could also be applied to identify LAB strains isolated from other food ecosystems.     

Polyphasic study of the genetic diversity of lactobacilli associated with 'Almagro' eggplants spontaneous fermentation, based on combined numerical analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoresis patterns

AIMS: The goal of this study was to assess the genetic diversity of lactic acid bacteria (LAB) from the complex natural ecosystem present in the spontaneous fermentation of 'Almagro' eggplants by a polyphasic approach based on molecular techniques. METHODS AND RESULTS: Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) were applied to 149 Lactobacillus isolates obtained from that fermentation process. Two random primers, OPL-05 and ArgDei-For, and two rare-cutting enzymes, SfiI and SmaI, chosen after preliminary testing on the basis of band intensity and distribution, were used. RAPD and PFGE generated electrophoretic patterns suitable for strain discrimination, but further discrimination was achieved when combined numerical analysis of the results from both methods and the results previously obtained by SDS-PAGE whole cell protein analysis, was carried out. The findings indicated a considerable degree of genomic diversity in the LAB microbiota studied and especially in the Lactobacillus plantarum isolates. In terms of species assignment, the polyphasic study allowed a definite and well-founded identification of 98.7% of the isolates. CONCLUSIONS: The combined numerical analysis of RAPD and PFGE patterns represented a useful tool to discriminate the diversity of the Lactobacillus strains responsible for the spontaneous fermentation of this pickle. The species identification and strain typing results from the polyphasic study were regarded as the most exact compromise yielding the fewest contradictions based on the available data. SIGNIFICANCE AND IMPACT OF THE STUDY: Combined numerical analysis of RAPD-PCR and PFGE patterns has not yet been employed to study the genetic diversity of LAB from an ecosystem like that found in fermenting vegetables.     

Genetic and phenotypic diversity of Saccharomyces sensu stricto strains isolated from Amarone wine

Individual yeast strains belonging to the Saccharomyces sensu stricto complex were isolated from Amarone wine produced in four cellars of the Valpolicella area (Italy) and characterized by conventional physiological tests and by RAPD-PCR and mtDNA restriction assays. Thirteen out of 20 strains were classified as Saccharomyces cerevisiae (ex S. cerevisiae p.r. cerevisiae and p.r. bayanus) and the remaining as Saccharomyces bayanus (ex S. cerevisiae p.r. uvarum). RAPD-PCR method proved to be a fast and reliable tool for identification of Saccharomyces sensu stricto strains and also gave intraspecific differentiation. Restriction analysis of mtDNA permitted to distinguish S. cerevisiae and S. bayanus species and to discern polymorphism among S. cerevisiae isolates. The assessment of the phenotypic diversity within the isolates by gas-chromatographic analysis of secondary fermentation products was explored. Small quantities of isobutanol were produced by most of the strains and higher amounts by some S. cerevisiae strains with phenotypes Gal^- and Mel^-; all S. bayanus strains produced low amounts of amilyc alcohols. From this study it appears that each winery owns particular strains, with different genetic and biochemical characteristics, selected by specific environmental pressures during the Amarone winemaking process carried out at low temperature in presence of high sugar content.     

Use of RAPD-PCR and TTGE for the evaluation of biodiversity of whey cultures for Grana Padano cheese

AIMS: This work was carried out in order to evaluate the microbial diversity of whey cultures collected from different Grana Padano cheese plants in Veneto region (north-east Italy) by means of RAPD-PCR and Temporal Temperature Gradient Gel Electrophoresis (TTGE) analysis. METHODS AND RESULTS: Lactobacillus helveticus was the dominant species among isolated thermophilic lactobacilli. RAPD-PCR with primers M13 and D8635 resulted a suitable method for typing Lact. helveticus at strain level. Thirteen different Lact. helveticus biotypes were detected in the seven whey cultures studied with one biotype present in all the whey cultures. Besides Lact. helveticus, Lact. delbrueckii subsp. lactis was the main microbial species detected by TTGE. CONCLUSIONS: RAPD-PCR resulted very useful in studying Lact. helveticus biodiversity; furthermore, TTGE analysis allowed to detect the dominant thermophilic microflora characteristic of Grana Padano cheese whey cultures. IMPACT OF THE STUDY: By the combined used of RAPD-PCR and TTGE it could be possible to follow the behaviour in strain or species composition of whey cultures during time.     

Study of the intra- and interlaboratory reproducibility of partial single base C-sequencing of the 16S rRNA gene and its applicability for the identification of members of the genus Streptococcus

The use of Single Base C-Sequencing of the first 500 bases of the 16S rRNA-gene (SBCS) combined with capillary electrophoresis was evaluated for the identification of reference strains of 30 different species within the genus Streptococcus. For SBCS, only dd-CTP's are used in the sequencing reactions instead of the four dideoxy bases and the primer is fluorescently labeled. The reproducibility, interlaboratory exchangeability and discriminative power of this method were studied by comparing the patterns obtained in three laboratories under highly standardized conditions. The interlaboratory reproducibility proved to be high, enabling the construction of a common database for the identification of strains belonging to the streptococcal species studied. Most of the examined species generated distinguishable profiles. SBCS did not differentiate between the closely related species S. constellatus and S. intermedius. Also S. thermophilus and S. vestibularis as well as S. mitis and S. pneumoniae showed highly resembling profiles. The previously reported heterogeneity within the species S. equinus was reflected by SBCS. For all other species, strains belonging to the same species generated indistinguishable patterns. In conclusion, Single Base C-sequencing of the first 500 bases of the 16S rRNA-gene could be a useful and widely applicable method for the identification of bacteria at the species level, with the added advantage of being more rapid and easier to automatize than full sequence determination.     

On the specificity of PCR detection of Listeria monocytogenes in food: a comparison of published primers

A total of nine pairs of primers, seven previously published and two newly developed, have been assayed for PCR detection of Listeria monocytogenes in food. They have been tested for specificity on a total of 72 strains including reference and food isolates belonging to L. monocytogenes and other species in the genus. First of all, a polyphasic approach has been carried out in order to establish a reference strain collection. They were biochemically and genetically characterized by API-Lis and randomly amplified polymorphic DNA PCR (RAPD-PCR), respectively. Random amplification of DNA was performed with M13, T7 and T3 universal primers and a data bank was created to compile the RAPD patterns of all the analyzed strains. The UPGMA cluster analysis of RAPD profiles with primer M13 showed eight clusters at 72.3% similarity. Clusters 2 and 7 corresponded to L. monocytogenes. Clusters 1 and 6 grouped L. ivanovii strains. Clusters 3, 4, 5 and 8 corresponded to L. grayi, L. innocua, L. welshimeri and L. seeligeri, respectively. Pattern analysis revealed the existence of miss-identified reference strains which was confirmed by 16S rDNA sequence analysis. RAPD-PCR is a rapid genetic test which helped to confirm strain identity. On the basis of PCR specificity results, primers LM1-LM2 were the best combination for the detection of L. monocytogenes since they only amplified the specific fragment in strains that had been genetically and biochemically assessed as belonging to the species. Specificity of other assayed primers is discussed.     

Evaluation of random amplified polymorphic DNA analysis and antibiotic susceptibility application in discrimination of salmonella typhimurium isolates in Iran

The purpose of this study was to determine the utility of RAPD-PCR and antibiotic susceptibility tests in differentiation of S. typhimurium isolates in Iran. Thirty isolates of S. typhimurium, selected based on animal source; place and time of the isolation and a reference strain of the bacterium were used in this study. Serotyping of the isolates was performed by reliable antisera and confirmed with a multiplex PCR. Genomic DNAs were extracted and subjected to an optimized RAPD-PCR, using two previously reported arbitrary primers (P1254 and 23L). Bacteria were also examined for resistance against 8 antibiotics, using a standard antibiotic susceptibility test. While the antibiotic susceptibility test resulted in the identification of 13 profiles of R-type among the bacterial isolates, application of primers P1254 and 23L in the RAPD-PCR could discriminate the isolates only in four and six profiles respectively. However, combination of the two methods could differentiate the 30 isolates in 20 different profiles. The results of this study indicate that the discriminatory power of RAPD-PCR for S. typhimurium is low but a combination of this method with antibiotic susceptibility test could be considered an easy and relatively reliable discriminatory approach in differentiation of S. typhimurium for epidemiologic purposes in Iran.     

Phenotypic and genotypic identification of yeasts from cheese

Eighty-five yeast strains isolated from different cheeses of Austria, Denmark, France, Germany, and Italy were identified using physiological methods and genotypically using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Good congruence was found between the phenotypic and genotypic data for 39 of the isolates. However, 26 isolates of Geotrichum could only be identified to the species level using the genotypic methods and 7 isolates were correctly identified to the genus level only using phenotypic identification methods. The phenotypic identification did not agree with the genotypic data for 14 yeast isolates. Using ubiquinone analysis, yeast cell wall sugars and the diazonium blue B test 5 incorrectly identified isolates with phenotypic methods could be identified genotypically. In addition the 7 isolates identified only to the genus level by the phenotypic methods and the 26 Geotrichum strains were identified to the species level using the polyphasic molecular approach mentioned above. Eleven strains remained unidentified. The 76 identified yeast isolates were assigned to 39 species, the most frequent assignments were made to Debaryomyces hansenii, Geotrichum candidum, Issatchenkia orientalis, Kluyveromyces lactis, K. marxianus, Saccharomyces cerevisiae, Yarrowia lipolytica, andCandida catenulata. It is proposed that Debaryomyces hansenii (Zopf) Lodder et Kreger-van Rij and Debaryomyces fabryi Ota should be reinstated. The RAPD-PCR data reinforced the view that the species Galactomyces geotrichum is heterogeneous with all of the Geotrichum isolates from cheese products being assigned G. geotrichum group A sensu M.T. Smith. It is suggested that the name Geotrichum candidum be conserved for this rather common species.     

Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera

Three molecular methods, RAPD-PCR analysis, electrophoretic karyotyping and RFLP of the PCR-amplified ITS regions (ITS1, ITS2 and the intervening 5.8S rDNA), were studied for accurate identification of Hanseniaspora and Kloeckera species as well as for determining inter- and intraspecific relationships of 74 strains isolated from different sources and/or geographically distinct regions. Of these three methods, PCR-RFLP analysis of ITS regions with restriction enzymes DdeI and HinfI is proposed as a rapid identification method to discriminate unambiguously between all six Hanseniaspora species and the single non-ascospore-forming apiculate yeast species Kloeckera lindneri. Electrophoretic karyotyping produced chromosomal profiles by which the seven species could be divided into four groups sharing similar karyotypes. Although most of the 60 strains examined exhibited a common species-specific pattern, a different degree of chromosomal-length polymorphism and a variable number of chromosomal DNA fragments were observed within species. Cluster analysis of the combined RAPD-PCR fingerprints obtained with one 10-mer primer, two microsatellite primers and one minisatellite primer generated clusters which with a few exceptions are in agreement with the groups as earlier recognized in DNA-DNA homology studies.     

Primer R108 performs best in the RAPD strain typing of threeAspergillus species frequently isolated from patients

We evaluated the suitability of various primers for the RAPD (random amplified polymorphic DNA) accurate species identification and strain typing of Aspergillus clinical isolates. Five primers described previously were tested for their discriminatory power in three Aspergillus species (A. fumigatus, A. niger agg. and A. flavus - 23 clinical isolates and 2 reference strains). Clustering of RAPD fingerprints corresponded well with the identification based on morphological features. All isolates were resolved as different strains using the primer R108 and the RAPD protocol optimized for a Robocycler thermal cycler. RAPD with the primer R108 thus can be considered to be a valuable, simple and powerful tool for identification and strain delineation of Aspergillus spp.     

Identification of lactobacilli isolated in traditional ripe wheat sourdoughs by using molecular methods

Eleven sourdoughs from Molise region (Southern-Italy) were subjected to microbiological analyses in order to select predominant lactobacilli species to be utilised as starter culture for bread production. A multiple approach was used, consisting of the growth in different culture media, DGGE analysis, 16S rRNA gene sequencing and RAPD-PCR typing. Forty-three lactobacilli were identified and four different species, facultatively or obligately heterofermentative lactobacilli, were found. Lactobacillus plantarum and Lactobacillus brevis represented the prevailing lactobacilli, while Lactobacillus casei and Lactobacillus paracasei ssp. paracasei were detected only in few samples. The use of different media demonstrated that there is no efficient medium for the study of sourdoughs and the cultivation in different substrates remains the best tool to obtain a picture of lactic acid bacteria population. DGGE and 16S rRNA gene sequencing allowed to obtain a reliable identification of strains, while RAPD-PCR resulted a suitable method for typing lactobacilli at strain level.