Molecular identification of vaginal lactobacilli isolated from Bulgarian women

Lactobacilli play an important role in maintaining the vaginal health of women. The development of suitable bacterial replacement therapies for the treatment of vaginosis requires knowledge of the vaginal lactobacilli species representation. The aim of this study was to identify at the species level vaginal Lactobacillus isolates obtained from Bulgarian women in childbearing age by using different molecular methods. Twenty-two strains of lactobacilli isolated from vaginal samples were identified and grouped according to their genetic relatedness. A combined approach, which included amplified ribosomal DNA restriction analysis (ARDRA), ribotyping and polymerase chain reaction (PCR) with species-specific oligonucleotide primers was applied. All vaginal isolates were grouped into 5 clusters in␣comparison with a set of 21 reference strains based␣on the initial ARDRA results, which was then confirmed by ribotyping. Finally, the strains were subjected to PCR analysis with eight different species-specific primer pairs, which allowed most of␣them to be classified as belonging to one of␣the␣following species: Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus plantarum. In conclusion, this study suggests that the most straightforward identification strategy for vaginal lactobacilli would be grouping by ARDRA or ribotyping, followed by PCR specific primers identification at species level.     

Vaginal lactic acid bacteria in the mare: evaluation of the probiotic potential of native Lactobacillus spp. and Enterococcus spp. strains

Lactic acid bacteria (LAB) are important members of the human vaginal microbiota and their presence is considered beneficial. However, little is known about native vaginal bacteria in other animal species such as the horse. The aim of this work was to quantify the vaginal lactic acid bacteria and lactobacilli of mares and to establish if selected equine vaginal lactic acid bacteria, particularly Lactobacillus and Enterococcus spp. strains, could exhibit potential as probiotics. The vaginal lactic acid bacteria and lactobacilli of 26 mares were quantified by plate counts. Five strains (three Lactobacillus spp. and two Enterococcus spp.) were characterised and adhesion to vaginal epithelial cells, antimicrobial activity and ability to form biofilms were evaluated. Lactic acid bacteria were recovered from the 26 samples and lactobacilli counts were detected in 18 out of 26 mares (69%). Probiotic properties tested in this study varied among the isolates and showed promising features for their use as equine probiotics.     

Taxonomic Lactobacillus Composition of Feces from Human Newborns during the First Few Days

Abstract Intestinal microbiota comprise a complex ecosystem whose equilibrium is crucial for the health of animal species. For humans, data exist on the microbiota composition in adult subjects, but few studies have addressed the microbiota composition in infants. In particular, data on the presence and species distribution of members of the genus Lactobacillus in newborns (less than one week old) are lacking. In the present work, the feces of healthy newborns were sampled to determine the taxonomic composition of Lactobacillus in the intestinal microbiota in a group of 16 neonates. In total, 1640 colony-forming units (CFU) were isolated, of which 420 grouped in the Lactobacillus genus by means of primary phenotypic characterization. The 420 isolates were further grouped into 125 strains on the basis of identical plasmid profiles. Of these 125 strains, 21 turned out to be permanent, i.e., they were identified in the feces of the same subject on several consecutive days. Sugar fermentation, DNA/DNA hybridization, and S-layer protein determination enabled us to classify 52 of the 125 strains as follows: L. paracasei (40 strains), L. delbrueckii sp. (1 strain), and L. acidophilus (sensu stricto) (11 strains). Based on the same criteria, the remaining 73 strains were tentatively allotted to the Johnson subgroup B, although hybridization experiments with probes specific for L. gasseri and L. johnsonii species were not performed. The presence of new species among these 73 strains cannot be excluded. Surprisingly, the obligately heterofermentative lactobacilli, L. reuteri in particular, were entirely absent from the feces of healthy newborns. Received: 26 March 1997; Accepted: 24 July 1997     

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

Phylogeny of tall fescue and related species using RFLPs

The wild species of tall fescue (Festuca arundinacea var.genuina Schreb.) represent a wide range of genetic variation and constitute potential germplasm for tall fescue improvement. Our objective was to evaluate genome specificity of the previously-identified DNA probes and to examine the phylogenetic relationship of tall fescue with six related species by using RFLP data. A total of 29 DNA probes from aPstI-genomic library of tall fescue were hybridized toEcoRI-orHindIII-digested DNA of 32 plants from sixFestuca species and fromLolium perenne L. Fifteen probes hybridized to all seven species. The remaining 14 probes showed differential hybridization patterns (i.e., ±), especially at the diploid and tetraploid levels. This hybridization pattern reflected genome divergence in these species. The DNA probes will be useful markers in breeding programs involving interspecific and intergeneric hybridization. Cluster analyses were performed using the average genetic distances calculated with the RFLP data from 53 probe-enzyme combinations. Generally, genotypes from the same species were grouped in the same cluster. These data indicated that tall fescue has a close relationship withF. pratensis Huds. (diploid),F. arundinacea var.glaucescens Boiss. (tetraploid), andL. perenne L. (diploid) and thatFestuca pratensis andL. perenne had the closest degree of relationship.This paper is a contribution of the Missouri Agricultural Experimental Station, Journal Series no. 11,798     

Tracing Streptococcus thermophilus strains in three-component yogurt starters

Three-component starters for yogurt were obtained on the base of starter LBB.BY 5-12 for traditional Bulgarian yogurt, containing strains Lactobacillus delbrueckii ssp. bulgaricus B5 and Streptococcus thermophilus A with the addition of either an exopolysaccharide-producing S. thermophilus strain 6V or the fast acidifying S. thermophilus strain N1. To differentiate between the three strains in the starter cultures, randomly amplified polymorphic DNA (RAPD) technique was applied to develop strain-specific probes. Southern hybridization against dot-blots of chromosomal DNA from the three S. thermophilus strains confirmed that two probes, derived from a 770 bp RAPD product obtained with primer RAPD-4 and a 290 bp sequence obtained with primer OPP-7 were specific for S. thermophilus 6V and S. thermophilus A, respectively, while no hybridization to S. thermophilus N1 DNA was observed. The selected probes were used to differentiate between S. thermophilus colonies on a solid agar medium by colony hybridization. The evaluation of the viable cell counts revealed that the populations of S. thermophilus A and the added S. thermophilus strains 6V or N1 in the three-component starters and in yogurt had nearly equal proportion allowing each strain to contribute to the enriched properties of starter and product.     

A DNA hybridization assay to identify toxic dinoflagellates in coastal waters: detection of Karenia brevis in the Rookery Bay National Estuarine Research Reserve

A DNA hybridization assay was developed in microtiter plate format to detect the presence of toxic dinoflagellates in coastal waters. Simultaneous detection of multiple species was demonstrated using Karenia brevis, Karenia mikimotoi, and Amphidinium carterae. Molecular probes were designed to detect both K. brevis and K. mikimotoi and to distinguish between these two closely related species. The assay was used to detect K. brevis in coastal waters collected from the Rookery Bay National Estuarine Research Reserve. Assay results were verified by species-specific PCR and sequence analysis. The presence/absence of K. brevis was consistent with microscopic observation. Assay sensitivity was sufficient to detect K. brevis in amounts defined by a regional monitoring program as “present” (≤1000 cells/L). The assay yielded quick colorimetric results, used a single hybridization temperature, and conserved the amount of genomic DNA utilized by employing one set of PCR primers. The microplate assay provides a useful tool to quickly screen large sample sets for multiple target organisms.     

Quantification of<Emphasis Type="Italic">Bacillus</Emphasis> species in a wastewater treatment system by the molecular analyses

Bacillus species were observed and quantified by molecular approaches, using the 165 rDNA primers/probes, in a wastewater treatment plant designed for the purpose of stimulating the growth ofBacillus species. The plant has been operating as a test plant since 1997 in the city of Ina, Japan, with excellent treatment performance. Observations byin situ hybridization, usingBacillus-specific probes, indicated thatBacillus strains were inhabited in the plant and their numbers decreased during the treatment process. Similar results were obtained from a quantitative PCR analysis using aBacillus-specific primer set, and the amount of DNA originating from variousBacillus species was maximally 1.91% of the total DNA in the wastewater treatment tank. Clone library analysis using theBacillus-specific primers suggested that, while the population was noticeably increased, the phylogenetic diversity of the increasingBacillus species was very low.     

Specific detection of Mycobacterium sp. genomic DNA using dual labeled gold nanoparticle based electrochemical biosensor

The present study was aimed at the development and evaluation of a DNA electrochemical biosensor for Mycobacterium sp. genomic DNA detection in a clinical specimen using a signal amplifier as dual-labeled AuNPs. The DNA electrochemical biosensors were fabricated using a sandwich detection strategy involving two kinds of DNA probes specific to Mycobacterium sp. genomic DNA. The probes of enzyme ALP and the detector probe both conjugated on the AuNPs and subsequently hybridized with target DNA immobilized in a SAM/ITO electrode followed by characterization with CV, EIS, and DPV analysis using the electroactive species para-nitrophenol generated by ALP through hydrolysis of para-nitrophenol phosphate. The effect of enhanced sensitivity was obtained due to the AuNPs carrying numerous ALPs per hybridization and a detection limit of 1.25 ng/ml genomic DNA was determined under optimized conditions. The dual-labeled AuNP-facilitated electrochemical sensor was also evaluated by clinical sputum samples, showing a higher sensitivity and specificity and the outcome was in agreement with the PCR analysis. In conclusion, the developed electrochemical sensor demonstrated unique sensitivity and specificity for both genomic DNA and sputum samples and can be employed as a regular diagnostics tool for Mycobacterium sp. monitoring in clinical samples.     

Novel SCAR primers for specific and sensitive detection of Agrobacterium vitis strains

An Agrobacterium vitis-specific DNA fragment (pAVS3) was generated from PCR polymorphic bands amplified by primer URP 2R. A. vitis specificity of this fragment was confirmed by Southern hybridization with genomic DNA from different Agrobacterium species. Sequence-characterized amplified region (SCAR) markers were developed for A. vitis specific detection, using 24-mer oligonucleotide primers designed from the flanking ends of the 670 bp insert in pAVS3. The SCAR primers amplified target sequences only from A. vitis strains and not from other Agrobacterium species or other bacterial genera. First round PCR detected bacterial cells between 5×10² and 1×10³ cfu/ml and the detection sensitivity was increased to as few as 2 cfu/ml by nested PCR. This PCR protocol can be used to confirm the potential presence of infectious A. vitis strains in soil and furthermore, can identify A. vitis strains from naturally infected crown galls.     

Development of Molecular Identification Method for Genus Alexandrium (Dinophyceae) Using Whole-Cell FISH

We have developed a method to identify species in the genus Alexandrium using whole-cell fluorescent in situ hybridization with FITC-labeled oligonucleotide probes that target large subunit ribosomal rRNA molecules. The probes were designed based on the sequence of the rDNA D1-D2 region of Alexandrium species. DNA probes specific for toxic A. tamarense and A. catenella and nontoxic A. affine, A. fraterculus, A. insuetum, and A. pseudogonyaulax, respectively, were applied to vegetative cells of all above Alexandrium species to test the sensitivity of the probes. Each DNA probe hybridized specifically with vegetative cells of the corresponding Alexandrium species and showed no cross-reactivity to noncorresponding Alexandrium species. In addition, no cross-reactivity of the probes was observed in experiments using concentrated natural seawater samples. The TAMAD2 probe, which is highly specific to A. tamarense, a common toxic species in Korean coastal waters, provides a simple and reliable molecular tool for identification of toxic Alexandrium species.     

Lactobacillus and Pediococcus species richness and relative abundance in the vagina of rhesus monkeys (Macaca mulatta)

Background The rhesus monkey is an important animal model to study human vaginal health to which lactic acid bacteria play a significant role. However, the vaginal lactic acid bacterial species richness and relative abundance in rhesus monkeys is largely unknown. Methods Vaginal swab samples were aseptically obtained from 200 reproductive‐aged female rhesus monkeys. Following Rogosa agar plating, single bacterial colonies representing different morphotypes were isolated and analyzed for whole‐cell protein profile, species‐specific polymerase chain reaction, and 16S rRNA gene sequence. Results A total of 510 Lactobacillus strains of 17 species and one Pediococcus acidilactici were identified. The most abundant species was Lactobacillus reuteri, which colonized the vaginas of 86% monkeys. Lactobacillus johnsonii was the second most abundant species, which colonized 36% of monkeys. The majority of monkeys were colonized by multiple Lactobacillus species. Conclusions The vaginas of rhesus monkeys are frequently colonized by multiple Lactobacillus species, dominated by L. reuteri.     

DNA homology between Candida albicans strains: Evidence to justify the synonymous status of C. stellatoidea

Genetic relatedness between strains of C. albicans and C. stellatoidea was studied by measuring G + C content and overall sequence homology. G + C contents determined by high-performance liquid chromatography were 32.6 to 34.2% for 26 strains of C. albicans and 33.0 to 33.9% for eight strains of C. stellatoidea. DNA-DNA hybridization with two C. albicans and two C. stellatoidea probes revealed that all 34 test strains formed a single cluster in which the extents of hybridization with the heterologous probes ranged between 77.9 and 105.6% of those with the homologous probes. These results give support to the unification of C. albicans and C. stellatoidea into a single species.     

Colorimetric detection of the toxic dinoflagellate Alexandrium minutum using sandwich hybridization in a microtiter plate assay

Rapid and reliable detection of harmful algae in coastal areas and shellfish farms is an important requirement for monitoring programs. Molecular technologies are rapidly improving the detection of phytoplankton and their toxins. Assays are based on the discrimination of genetic differences in the species. A commercially available PCR ELISA Dig Detection Kit in a microtiter plate was adapted for the rapid assessment of specificity of the two probes used in a sandwich hybridization assay. The toxic dinoflagellate Alexandrium minutum was used as the target organism and a capture and signal probe were designed for a species-specific identification of this species. This assay also provided the necessary specificity tests prior to the probes being adapted to an automated biosensor using a sandwich hybridization format. All probes regardless of the detection method must be extensively tested prior to use in the field. Total rRNA was isolated from three different strains of A. minutum and the mean concentration of RNA per cell of was determined to be 0.028 ng ± 0.003. Thus, a standard calibration curve for different RNA concentrations was determined so that cell numbers could be inferred from the assay. The assay and the standard curve were evaluated by using spiked field samples. The results demonstrated that the molecular assay was able to detect A. minutum cells at different cell counts in the presence of a complex background.     

Ostracodology in time and space: looking back on fifteen International Symposia on Ostracoda,and the times in between

Nuclease protection assay (NPA) probes were designed to target the rRNA of Chaetoceros curvisetus and Skeletonema costatum, and quantitative sandwich hybridization integrated with nuclease protection assay (NPA-SH) was developed to detect C. curvisetus and S. costatum in culture and field samples in Jiaozhou Bay, China. The specificity and validity of the NPA-SH technique were tested with cultured pure strains, mixed strains and field samples, and by comparison with that of microscopy observation. The linear detection range for C. curvisetus was 4.2 × 10⁴ to 1.2 × 10⁶ cells with a detection limit of 42 cells ml⁻¹. The linear range for S. costatum was 6.0 × 10⁴ to 1.0 × 10⁶ cells with a detection limit of 60 cells ml⁻¹. The NPA-SH in this study provides a convenient tool for rapid assessment of HAB species in marine environments. Handling editor: D. Hamilton     

Relationships betweenOryza species (Poaceae) based on 5S DNA sequences

Relationships between 9Oryza species, covering 6 different genomes, have been studied using hybridization and nucleotide sequence information from the5S Dna locus. Four to five units of the major size class of 5S DNA in each species, 55 units in all, were cloned and sequenced. Both hybridization and sequence data confirmed the basic differences between the A and B, C, D genome species suggested by morphological and cytological data. The 5S DNA units of the A genome species were very similar, as were the ones from the B, C, and D genome-containing species. The 5S DNA ofO. australiensis (E genome) grouped with the B, C, D cluster, while the units ofO. brachyantha (F genome) were quite different and grouped away from all other species. 5S DNA units fromO. minuta, O. latifolia, O. australiensis, andO. brachyantha hybridized strongly, and preferentially, to the genomic DNA from which the units were isolated and hence could be useful as species/genome specific probes. The 5S DNA units fromO. sativa, O. nivara, andO. rufipogon provided A genome-specific probes as they hybridized preferentially to A genome DNA. The units fromO. punctata andO. officinalis displayed weaker preferential hybridization toO. punctata DNA, possibly reflecting their shared genome (C genome).     

Detection of Hyperthermophilic Archaea of the Genus Desulfurococcus by Hybridization with Oligonucleotide Probes

Based on the analysis of nucleotide sequences of 16S rRNA, oligonucleotide probes were designed for the detection and identification of representatives of the genus Desulfurococcus (kingdom Crenarchaeota of the domain Archaea). The detection procedure included obtaining PCR products on DNA isolated from pure cultures, enrichments, or natural samples with a designed Crenarchaeota-specific primer pair: Cren 7F (5"-TTCCGGTTGATCCYGCCGGACC-3") and Cren 518R (5"-GCTGGTWTTACCGCGGCGGCTGA-3"). The PCR products were hybridized with Dig-11-dUTP–labeled oligonucleotide probes targeting the genus Desulfurococcus (Dco 198, 5"-CGTTAACYCCYGCCACACC-3") and its species D. mobilis (Dco_mob 198, 5"-CGTTAACCCCTGCCACACC-3") and D. amylolyticus (Dco_amy 198, 5"-CGTTAACCCCCGCCACACC-3"). With the use of these primers and probes, four new strains isolated from hydrotherms of Kamchatka and Kunashir Island were identified as members of the speciesDesulfurococcus amylolyticus. Desulfurococcus representatives were detected in several natural samples, including a sample taken from a marine hydrotherm at Kunashir Island; this demonstrates that representatives of this genus occur not only in terrestrial but also in marine environments.     

Centromeric repetitive DNA sequences in the genus Brassica

Representatives of two major repetitive DNA sequence families from the diploid Brassica species B. campestris and B. oleracea were isolated, sequenced and localized to chromosomes by in situ hybridization. Both sequences were located near the centromeres of many chromosome pairs in both diploid species, but major sites of the two probes were all on different chromosome pairs. Such chromosome specificity is unusual for plant paracentromeric repetitive DNA. Reduction of stringency of hybridization gave centromeric hybridization sites on more chromosomes, indicating that there are divergent sequences present on other chromosomes. In tetraploid species derived from the diploids, the number of hybridization sites was different from the sum of the diploid ancestors, and some chromosomes had both sequences, indicating relatively rapid homogenization and copy number evolution since the origin of the tetraploid species.     

Assessment of the degree of restriction fragment length polymorphism in Brassica

Summary The feasibility of creating a restriction fragment length polymorphism (RFLP) linkage map in Brassica species was assessed by screening EcoRI-, HindIII-, or EcoRV-digested total genomic DNA from several accessions of B. campestris, B. oleracea, and B. napus using random genomic DNA clones from three Brassica libraries as hybridization probes. Differences in restriction fragment hybridization patterns occurred at frequencies of 95% for comparisons of accessions among species, 79% for comparisons of accessions among subspecies within species, and 70% for comparisons among accessions within subspecies. In addition, species differences in the level of hybridization were noted for some clones. The high degree of polymorphism found even among closely related Brassica accessions indicates that RFLP analysis will be a very useful tool in genetic, taxonomic, and evolutionary studies of the Brassica genus. Development of RFLP linkage maps is now in progress.