Proposal of Henriciella barbarensis sp. nov. and Henriciella algicola sp. nov., stalked species of the genus and emendation of the genus Henriciella.

Two Gram-negative, heterotrophic, aerobic, prosthecated, marine bacteria, designated strains MCS23T and MCS27T, were isolated from seawater samples. NaCl was required for growth. The major polar lipid detected in strain MCS27T was phosphatidylglycerol, whereas those detected in MCS23T were phosphatidylglycerol, sulfoquinovosyl diacylglycerol and 1,2-diacyl-3-α-d-glucuronopyranosyl-sn-glycerol taurineamide. The most abundant cellular fatty acids were C18 : 1ω7 and C16 : 0, hydroxyl-fatty acids were 3-OH C12 : 0 in both strains and 3-OH C11 : 0 in MCS23T. Strains MCS23T and MCS27T had DNA G+C contents of 57.0 and 55.0 mol%, respectively. The two strains shared 99.3 % 16S rRNA gene sequence similarity; levels of similarity with the type strains of species of the genus Henriciella were 99.4-97.8 % but DNA-DNA hybridizations were 53 % or lower. Besides their 16S rRNA gene sequences, the novel strains can be differentiated from other species of the genus Henriciella by cell morphology, lipid and fatty acid patterns and enzyme activities. The data obtained led to the identification of two novel species, for which the names Henriciella barbarensis sp. nov. (type strain MCS23T=LMG 28705T=CCUG 66934T) and Henriciella algicola sp. nov. (type strain MCS27T=LMG 29152T=CCUG 67844T) are proposed. As these two novel species are the first prosthecate species in the genus Henriciella, an emended genus description is also provided.

Cauliform bacteria lacking phospholipids from an abyssal hydrothermal vent: proposal of Glycocaulis abyssi gen. nov., sp. nov., belonging to the family Hyphomonadaceae.

Cauliform bacteria are prosthecate bacteria often specialized for oligotrophic environments. A polyphasic approach, comprising 16S rRNA gene sequencing, lipid analysis and salt tolerance characterizations, was used to clarify the taxonomy of one isolate, strain MCS 33(T), obtained from above the hot water plume of a deep-sea hydrothermal vent near Vancouver island, Canada. Cells contained no detectable phospholipids or sulpholipids, but did contain 1,2-di-O-acyl-3-O-α-D-glucopyranosylglycerol, 1,2-di-O-acyl-3-O-α-D-glucopyranuronosylglycerol and the novel lipid 1,2-di-O-acyl-3-[O-α-D-glucopyranuronosyl]glycerol-6'-N-glycine. It is assumed that the various glucoronosyl lipids are replacing, at least partially, the phospholipids in their various tasks in the cell cycle. The G+C content of the genomic DNA of strain MCS 33(T) was 62.8 mol%, and Q10 was the predominant respiratory ubiquinone. The 16S rRNA gene sequence of this chemoheterotrophic, aerobic, moderately halophilic strain showed only a low similarity of 94.4% to that of Oceanicaulis alexandrii C116-18(T), and both strains also differed based on their lipids. Although the novel strain was isolated from seawater sampled near a hydrothermal vent, its optimum temperature for growth was 30 °C. The main cellular fatty acids were C18:1ω7c, C18:0 and the unknown fatty acid ECL 11.798, and the main hydroxy fatty acid was C12:0 3-OH. The strain is proposed to represent a novel species of a new genus, Glycocaulis abyssi gen. nov., sp. nov. The type strain of the type species is MCS 33(T) (=LMG 27140(T)=CCUG 62981(T)).

Prosthecate sphingomonads: proposal of Sphingomonas canadensis sp. nov.

Two stalked, aerobic, catalase- and oxidase-positive rod-shaped isolates, VKM B-1508 ( = CB 258) and FWC47(T), were analysed using a polyphasic approach. While the morphology and the 16S rRNA gene sequence of strain VKM B-1508 were 100% identical to the ones of Sphingomonas leidyi DSM 4733(T), the morphology of FWC47(T) was different, and the closest recognized species were Sphingomonas oligophenolica S213(T) ( = DSM 17107(T)) and Sphingomonas leidyi DSM 4733(T) with 97.2% and 97.0% 16S rRNA gene sequence similarity, respectively. DNA-DNA hybridization studies supported the differentiation of strain FWC47(T) from S. oligophenolica and S. leidyi. Strain FWC47(T) grew optimally at 28-30 °C, and pH 6.0-8.0. The major respiratory quinone was Q10 and the major polyamine was sym-homospermidine. The major fatty acids were C(17:1)ω6c and C(18:1)ω7c and C(15:0) 2-OH was the major 2-hydroxy fatty acid. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidyldimethylethylamine and unidentified sphingoglycolipids. The G+C content of the genomic DNA of strain FWC47(T) was 67.1 mol%. Strain FWC47(T) differed from S. leidyi by its ability to assimilate l-alanine, maltose and sucrose, by the presence of ß-galactosidase and α-chymotrypsin, and the lack of valine arylamidase and ß-glucosidase activities. Contrary to S. leidyi, FWC47(T) did not reduce nitrate and could not use fructose, acetate and N-acetyl-glusosamine. In the genus Sphingomonas, the dimorphic life cycle involving a prosthecate sessile and a flagellated swarmer cell was hitherto only known from Sphingomonas leidyi. Therefore, strain FWC47(T) represents an additional distinct prosthecate species in this genus for which the name Sphingomonas canadensis is proposed. The type strain is FWC47(T) ( =LMG 27141(T) =CCUG 62982(T)).

Surface microbial consortia from Livarot, a French smear-ripened cheese.

The surface microflora (902 isolates) of Livarot cheeses from three dairies was investigated during ripening. Yeasts were mainly identified by Fourier transform infrared spectroscopy. Geotrichum candidum was the dominating yeast among 10 species. Bacteria were identified using Biotype 100 strips, dereplicated by repetitive extragenic palindromic PCR (rep-PCR); 156 representative strains were identified by either BOX-PCR or (GTG)(5)-PCR, and when appropriate by 16S rDNA sequencing and SDS-PAGE analysis. Gram-positive bacteria accounted for 65% of the isolates and were mainly assigned to the genera Arthrobacter , Brevibacterium , Corynebacterium , and Staphylococcus . New taxa related to the genera Agrococcus and Leucobacter were found. Yeast and Gram-positive bacteria strains deliberately added as smearing agents were sometimes undetected during ripening. Thirty-two percent of the isolates were Gram-negative bacteria, which showed a high level of diversity and mainly included members of the genera Alcaligenes , Hafnia , Proteus , Pseudomonas , and Psychrobacter . Whatever the milk used (pasteurized or unpasteurized), similar levels of biodiversity were observed in the three dairies, all of which had efficient cleaning procedures and good manufacturing practices. It appears that some of the Gram-negative bacteria identified should now be regarded as potentially useful in some cheese technologies. The assessment of their positive versus negative role should be objectively examined.

Salinimicrobium marinum sp. nov., a halophilic bacterium of the family Flavobacteriaceae, and emended descriptions of the genus Salinimicrobium and Salinimicrobium catena.

Two novel heterotrophic, facultatively anaerobic, gliding and yellow-pigmented bacteria, designated strains KMM 6270(T) and KMM 6320, were isolated from different marine environments and studied using a polyphasic taxonomic approach. 16S rRNA gene sequence analysis placed the strains within the family Flavobacteriaceae. Strains KMM 6270(T) and KMM 6320 were most closely related to the type strains of recognized species of the genus Salinimicrobium (95.0-96.6 % 16S rRNA gene sequence similarity). The G+C content of the genomic DNA was 40-41 mol%. The strains grew with 0.5-15 % (w/v) NaCl (optimum 4 % NaCl) and at 4-41 °C (optimum 28-32 °C). Aesculin and gelatin were hydrolysed, but agar, casein, DNA and chitin were not. The phylogenetic data taken together with the results of the genotypic and phenotypic studies permit the classification of strains KMM 6270(T) and KMM 6320 as members of a novel species of the genus Salinimicrobium, for which the name Salinimicrobium marinum sp. nov. is proposed. The type strain is KMM 6270(T) (=KCTC 12719(T)=LMG 25395(T)).

Taxonomic structure and stability of the bacterial community in belgian sourdough ecosystems as assessed by culture and population fingerprinting.

A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.

Commercial ripening starter microorganisms inoculated into cheese milk do not successfully establish themselves in the resident microbial ripening consortia of a South german red smear cheese.

Production of smear-ripened cheese critically depends on the surface growth of multispecies microbial consortia comprising bacteria and yeasts. These microorganisms often originate from the cheese-making facility and, over many years, have developed into rather stable, dairy-specific associations. While commercial smear starters are frequently used, it is unclear to what degree these are able to establish successfully within the resident microbial consortia. Thus, the fate of the smear starters of a German Limburger cheese subjected to the "old-young" smearing technique was investigated during ripening. The cheese milk was supplemented with a commercial smear starter culture containing Debaryomyces hansenii, Galactomyces geotrichum, Arthrobacter arilaitensis, and Brevibacterium aurantiacum. Additionally, the cheese surface was inoculated with an extremely stable in-house microbial consortium. A total of 1,114 yeast and 1,201 bacterial isolates were identified and differentiated by Fourier transform infrared spectroscopy. Furthermore, mitochondrial DNA restriction fragment length polymorphism, random amplified polymorphic DNA, repetitive PCR, and pulsed field gel electrophoresis analyses were used to type selected isolates below the species level. The D. hansenii starter strain was primarily found early in the ripening process. The G. geotrichum starter strain in particular established itself after relocation to a new ripening room. Otherwise, it occurred at low frequencies. The bacterial smear starters could not be reisolated from the cheese surface at all. It is concluded that none of the smear starter strains were able to compete significantly and in a stable fashion against the resident microbial consortia, a result which might have been linked to the method of application. This finding raises the issue of whether addition of starter microorganisms during production of this type of cheese is actually necessary.

Genotypic diversity, antimicrobial resistance, and virulence factors of human isolates and probiotic cultures constituting two intraspecific groups of Enterococcus faecium isolates.

The intraspecific relationships among a collection of Enterococcus faecium isolates comprising probiotic cultures and human clinical isolates were investigated through the combined use of two high-resolution DNA-fingerprinting techniques. In addition, the incidences of antimicrobial resistance and virulence traits were investigated. A total of 128 E. faecium isolates from human clinical or nonclinical sources or used as probiotic cultures were subjected to fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting and pulsed-field gel electrophoresis (PFGE) analysis of SmaI macrorestriction patterns. Susceptibilities to 16 antimicrobial agents were tested using broth microdilution, and the presence of the corresponding resistance genes was investigated using PCR. Multiplex PCR was used to detect the presence of the enterococcal virulence genes asa1, gelE, cylA, esp, and hyl. The results of the study showed that two intraspecific genomic groups (I and II) were obtained in FAFLP analysis. PFGE analysis demonstrated high variability within these two groups but also indicated that some probiotic cultures were indistinguishable and that a number of clinical isolates may be reisolations of commercial probiotic cultures. Compared to group II, which contained the majority of the probiotic isolates and fewer human clinical isolates, higher phenotypic and genotypic resistance frequencies were observed in group I. Two probiotic isolates were phenotypically resistant to erythromycin, one of which contained an erm(B) gene that was not transferable to enterococcal recipients. None of the probiotic E. faecium isolates demonstrated the presence of the tested virulence genes. The previously reported observation that E. faecium consists of two intraspecific genomic groups was further substantiated by FAFLP fingerprinting of 128 isolates. In combination with antimicrobial resistance and virulence testing, this grouping might represent an additional criterion in assessing the safety of new potential probiotic E. faecium isolates.

Phylogeny and identification of Pantoea species associated with plants, humans and the natural environment based on multilocus sequence analysis (MLSA).

Species belonging to the genus of Pantoea are commonly isolated from plants, humans and the natural environment. The species of the genus are phenotypically closely related, making rapid identification of Pantoea strains to the species level difficult. Multilocus sequence analysis (MLSA) was evaluated as a means for rapid classification and identification of Pantoea strains. Four housekeeping genes, gyrB, rpoB, atpD and infB, were sequenced for strains assigned to the genus. Included in the study were (1) reference strains from the seven currently recognized species of Pantoea, (2) strains belonging to Brenner DNA groups II, IV and V, previously isolated from clinical samples and difficult to identify because of high phenotypic similarity to P. agglomerans or P. ananatis and (3) isolates from diseased Eucalyptus, maize and onion, assigned to the genus on the basis of phenotypic tests. Phylogenetic trees were constructed from the sequences of the four housekeeping genes. The "core"Pantoea species formed a cluster separate from the "Japanese" species which formed a tight cluster that included the genus Tatumella when the tree was based on concatenated sequences of the four genes. The MLSA data further suggested the existence of ten potential novel species, phylogenetically related to the currently recognized Pantoea species and the possible inclusion of Pectobacterium cypripedii in the genus Pantoea. When compared with DNA-DNA hybridization data, a good congruence was observed between both methods, with gyrB sequence data being the most consistent. In conclusion, MLSA of partial nucleotide sequences of the genes gyrB, rpoB, atpD and infB can be used for classification, identification and phylogenetic analyses of Pantoea strains.

alpha-Chitinase activity among lactic acid bacteria.

Chitin is a polysaccharide widely distributed in nature. Among 115 strains from 29 species of lactic acid bacteria only strains belonging to Carnobacterium divergens and Carnobacterium maltaromaticum hydrolyzed alpha-chitin. This activity was not affected by temperature (10 degrees C versus 30 degrees C) and in most cases not subject to glucose catabolite repression.

Validation of the (GTG)(5)-rep-PCR fingerprinting technique for rapid classification and identification of acetic acid bacteria, with a focus on isolates from Ghanaian fermented cocoa beans.

Amplification of repetitive bacterial DNA elements through the polymerase chain reaction (rep-PCR fingerprinting) using the (GTG)(5) primer, referred to as (GTG)(5)-PCR fingerprinting, was found a promising genotypic tool for rapid and reliable speciation of acetic acid bacteria (AAB). The method was evaluated with 64 AAB reference strains, including 31 type strains, and 132 isolates from Ghanaian, fermented cocoa beans, and was validated with DNA:DNA hybridization data. Most reference strains, except for example all Acetobacter indonesiensis strains and Gluconacetobacter liquefaciens LMG 1509, grouped according to their species designation, indicating the usefulness of this technique for identification to the species level. Moreover, exclusive patterns were obtained for most strains, suggesting that the technique can also be used for characterization below species level or typing of AAB strains. The (GTG)(5)-PCR fingerprinting allowed us to differentiate four major clusters among the fermented cocoa bean isolates, namely A. pasteurianus (cluster I, 100 isolates), A. syzygii- or A. lovaniensis-like (cluster II, 23 isolates), and A. tropicalis-like (clusters III and IV containing 4 and 5 isolates, respectively). A. syzygii-like and A. tropicalis-like strains from cocoa bean fermentations were reported for the first time. Validation of the method and indications for reclassifications of AAB species and existence of new Acetobacter species were obtained through 16S rRNA sequencing analyses and DNA:DNA hybridizations. Reclassifications refer to A. aceti LMG 1531, Ga. xylinus LMG 1518, and Ga. xylinus subsp. sucrofermentans LMG 18788(T).

Infectivity of Lactobacillus rhamnosus and Lactobacillus paracasei isolates in a rat model of experimental endocarditis.

The potential pathogenicity of selected (potentially) probiotic and clinical isolates of Lactobacillus rhamnosus and Lactobacillus paracasei was investigated in a rat model of experimental endocarditis. In addition, adhesion properties of the lactobacilli for fibrinogen, fibronectin, collagen and laminin, as well as the killing activity of the platelet-microbicidal proteins fibrinopeptide A (FP-A) and connective tissue activating peptide 3 (CTAP-3), were assessed. The 90 % infective dose (ID(90)) of the L. rhamnosus endocarditis isolates varied between 10(6) and 10(7) c.f.u., whereas four of the six (potentially) probiotic L. rhamnosus isolates showed an ID(90) that was at least 10-fold higher (10(8) c.f.u.) (P<0.001). In contrast, the two other probiotic L. rhamnosus isolates exhibited an ID(90) (10(6) and 10(7) c.f.u.) comparable to the ID(90) of the clinical isolates of this species investigated (P>0.05). Importantly, these two probiotic isolates shared the same fluorescent amplified fragment length polymorphism cluster type as the clinical isolate showing the lowest ID(90) (10(6) c.f.u.). L. paracasei tended to have a lower infectivity than L. rhamnosus (ID(90) of 10(7) to > or =10(8) c.f.u.). All isolates had comparable bacterial counts in cardiac vegetations (P>0.05). Except for one L. paracasei strain adhering to all substrates, all tested lactobacilli adhered only weakly or not at all. The platelet peptide FP-A did not show any microbicidal activity against the tested lactobacilli, whereas CTAP-3 killed the majority of the isolates. In general, these results indicate that probiotic lactobacilli display a lower infectivity in experimental endocarditis compared with true endocarditis pathogens. However, the difference in infectivity between L. rhamnosus endocarditis and (potentially) probiotic isolates could not be explained by differences in adherence or platelet microbicidal protein susceptibility. Other disease-promoting factors may exist in these organisms and warrant further investigation.

Identification of lactic acid bacteria isolated from human blood cultures.

Fifteen lactic acid bacterial strains were isolated from blood cultures from 15 different patients in the Faculty Hospital in Brno, Czech Republic. All strains were identified using biochemical tests and repetitive PCR using the (GTG)5 primer. Doubtful identification results were confirmed by whole-cell protein analysis. The strains were assigned to the genera Lactobacillus (eight strains representing seven species), Leuconostoc (six strains representing four species) and Weissella (one strain). Antibiotic susceptibility testing was performed using the E-test and revealed high-level resistance to cotrimoxazol, metronidazole, vancomycin and teicoplanin, but nearly all strains were susceptible to erythromycin, clindamycin, ampicillin and penicillin.

A FAFLP system for the improved identification of plant-pathogenic and plant-associated species of the genus Pantoea.

The majority of Pantoea species are either plant-pathogenic or plant-associated and cause a wide variety of symptoms on a range of hosts. Identification of Pantoea species is difficult due to minor differences in phenotypic characteristics between them and related Enterobacteriaceae. Fluorescent amplified fragment length polymorphism (FAFLP) analysis was investigated for use as a rapid, molecular-based identification technique to the species level of the genus Pantoea. Following analysis of the band patterns generated by FAFLP, seven distinct clusters were observed, one for each validly published species of the genus. FAFLP has proven to be a rapid, reproducible identification technique for all species of the genus Pantoea.

Enterococcus and Lactobacillus contamination of raw milk in a farm dairy environment.

Enterococci and lactobacilli are ubiquitously found in the intestinal microflora of humans and animals. The aim of the present study was to determine the importance of bovine faeces as a source of these organisms in raw milk. One hundred and fifty six putative enterococci and 362 lactobacilli were isolated from bovine faeces (n=26), cows' teats, raw milk, the milking machine and the milking environment on one farm. The clonal relationships of each group were investigated using Pulsed-Field Gel Electrophoresis and representatives of the different clusters were identified by repetitive DNA element (rep)-PCR fingerprinting, protein profiling, phenylalanyl-tRNA synthase (pheS) sequence analysis or 16S rDNA gene sequencing. Lactobacilli were present at approximately 3 orders of magnitude greater than enterococci in the bovine faeces. The majority of the bovine faecal enterococcal isolates were identified as Aerococcus viridans. Seven teat isolates belonged to a potential novel Aerococcus sp. and one bovine faecal isolate to a potential second novel Aerococcus sp. The lactobacilli present in the bovine faeces were predominantly Lactobacillus mucosae and Lactobacillus brevis, with small numbers of Lactobacillus plantarum. Only one Enterococcus (a strain of E. casseliflavus) out of 76 and one Lactobacillus (a strain of L. parabuchneri/kefir) out of 247 of the bovine faecal isolates was found in the milk. The major source of these bacteria in the milk was the milking equipment.

Accuracy of species identity of commercial bacterial cultures intended for probiotic or nutritional use.

Independent studies have indicated that the microbiological composition of several commercial probiotic products does not correspond to the product label information. The present study set out to investigate to what extent these problems may be due to the use of misidentified cultures at the onset of production. For this purpose, 213 cultures of lactic acid bacteria (LAB) and propionibacteria intended for probiotic or nutritional use were collected from 26 manufacturers of probiotic products, three international culture collections and one research institute. The accuracy of the taxonomic identity provided by the strain depositor was assessed through a polyphasic approach based on validated and standardized identification methods including fluorescent amplified fragment length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, protein profiling and partial 16S rDNA sequencing. The majority of the cultures were received as members of the genera Lactobacillus (57%) and Bifidobacterium (22%); however, propionibacteria, enterococci, Lactococcus lactis (subsp. lactis), Streptococcus thermophilus and pediococci were also obtained. Upon reidentification, 46 cases of misidentification at the genus level (n=19) or species level (n=27) were recorded, including 34 commercial probiotic cultures deposited by 10 different companies. The finding that more than 28% of the commercial cultures intended for human and/or animal probiotic use were misidentified at the genus or species level suggests that many cases of probiotic product mislabeling originate from the incorporation of incorrectly identified strains. A large number of these discrepancies could be related to the use of methods with limited taxonomic resolution (e.g., API strips) or that are unsuitable for reliable identification up to species level (e.g., pulsed-field gel electrophoresis and randomly amplified polymorphic DNA analysis). The current study has again highlighted that reliable identification of LAB and propionibacteria requires molecular methods with a high taxonomic resolution that are linked to up-to-date identification libraries.

Mining fatty acid databases for detection of novel compounds in aerobic bacteria.

This study examines how the discriminatory power of an automated bacterial whole-cell fatty acid identification system can be significantly enhanced by exploring the vast amounts of information accumulated during 15 years of routine gas chromatographic analysis of the fatty acid content of aerobic bacteria. Construction of a global peak occurrence histogram based upon a large fatty acid database is shown to serve as a highly informative tool for assessing the delineation of the naming windows used during the automatic recognition of fatty acid compounds. Along the lines of this data mining application, it is suggested that several naming windows of the Sherlock MIS TSBA50 peak naming method may need to be re-evaluated in order to fit more closely with the bulk of observed fatty acid profiles. At the same time, the global peak occurrence histogram has put forward the delineation of 32 new peak naming windows, accounting for a 26% increase in the total number of fatty acid features taken into account for bacterial identification. By scrutinizing the relationships between the newly delineated naming windows and the many taxonomic units covered within a proprietary fatty acid database, all new naming windows were proven to correspond with stable features of some specific groups of microorganisms. This latter analysis clearly underscores the impact of incorporating the new fatty acid compounds for improving the resolution of the bacterial identification system and endorses the applicability of knowledge discovery in databases within the field of microbiology.

Biodiversity of lactic acid bacteria in Romanian dairy products.

Traditionally fermented dairy products are still a very important part of the daily food in Romania, especially for people living in the countryside. To study the biodiversity of lactic acid bacterium strains of these products, 110 samples (raw and fermented milk, sour cream, and cheese) were collected from farm houses, monasteries, and local markets throughout Romania. Lactic acid bacteria (LAB) were isolated using six different cultivation conditions. All 599 isolates were tested for their Gram reaction, catalase activity, and morphology. A rep-PCR fingerprinting technique with the (GTG)5 primer and, in some cases SDS-PAGE of total cell proteins and 16S rRNA gene sequencing were used to cluster and/or identify the LAB. The biodiversity of the isolated strains was correlated with the type of product and/or technology applied. The most frequent LAB found in Romanian raw milk and fermented dairy products were Lactococcus lactis, Leuconostoc spp., and Enterococcus spp. Among the latter, a new species E. saccharominimus was found.

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.

Grouping of streptomycetes using 16S-ITS RFLP fingerprinting.

A total of 463 Streptomyces and Kitasatospora type strains were screened using 16S-ITS RFLP fingerprinting (combined restriction digest using enzymes BstUI and HaeIII). In total, 59 clusters could be delineated, each comprising multiple strains with nearly identical patterns. Good correlation was found in general with phylogeny, as revealed by 16S rDNA sequencing. Most strains assigned to a particular 16S-ITS RFLP cluster were classified into the corresponding 16S sequencing cluster whether a 16S similarity cut-off value of 97 or 98% was used. We conclude that the taxonomic resolution of 16S-ITS RFLP fingerprinting is higher than that of 16S rDNA sequencing; this may provide a tool for reducing the number of laborious DNA-DNA hybridizations necessary for discovering potentially new species within Streptomyces.