Differentiation of Lactobacillus delbrueckii subspecies by ribotyping and amplified ribosomal DNA restriction analysis (ARDRA)

AIMS: To differentiate the subspecies of Lactobacillus delbrueckii, subsp. delbrueckii, subsp. lactis and subsp. bulgaricus. METHODS AND RESULTS: Amplified ribosomal DNA restriction analysis (ARDRA) and ribotyping were applied to over 30 strains. Both methods analyse the ribosomal genes which carry useful information about the evolutionary and taxonomic relationship among bacteria. The methods proved to be reliable and highly reproducible. ARDRA was applied to 16S rDNA, 23S rDNA and the IGS region, thus covering the whole rrn operon with eight restriction enzymes. Only EcoRI differentiated Lact. delbrueckii subsp. bulgaricus from Lact. delbrueckii subsp. delbrueckii/Lact. delbrueckii subsp. lactis, which confirmed the finding of other authors. Ribotyping with different enzymes under precisely optimized conditions revealed a high level of strain polymorphism. Only ribotyping with EcoRI allowed differentiation of the three subspecies on the basis of typical hybridization patterns. CONCLUSION: The successful differentiation of the three subspecies of Lact. delbrueckii by EcoRI ribotyping offers a new possibility for precise identification and differentiation of strains and new isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Both methods could be used for differentiation of Lact. delbrueckii subspecies.     

Evaluation of amplified ribosomal DNA restriction analysis (ARDRA) and species-specific PCR for identification of Bifidobacterium species

Molecular biological methods based on genus-specific PCR, species-specific PCR, and amplified ribosomal DNA restriction analysis (ARDRA) of two PCR amplicons (523 and 914bp) using six restriction enzymes were used to differentiate among species of Bifidobacterium. The techniques were established using DNA from 16 type and reference strains of bifidobacteria of 11 species. The discrimination power of 914bp amplicon digestion was higher than that of 523bp amplicon digestion. The 914bp amplicon digestion by six restrictases provided unique patterns for nine species; B. catenulatum and B. pseudocatenulatum were not differentiated yet. The NciI digestion of the 914bp PCR product enabled to discriminate between each of B. animalis, B. lactis, and B. gallicum. The reference strain B. adolescentis CCM 3761 was reclassified as a member of the B. catenulatum/B. pseudocatenulatum group. The above-mentioned methods were applied for the identification of seven strains of Bifidobacterium spp. collected in the Culture Collection of Dairy Microorganisms (CCDM). The strains collected in CCDM were differentiated to the species level. Six strains were identified as B. lactis, one strain as B. adolescentis.     

Rapid amplified ribosomal DNA restriction analysis (ARDRA) identification of Lactobacillus spp. isolated from fecal and vaginal samples

Amplified ribosomal DNA restriction analysis (ARDRA) was used to identify different species of Lactobacillus isolated from human faeces and vagina. PCR-ARDRA was performed using a set of four restriction enzymes, able to differentiate fourteen species of Lactobacillus. The PCR-ARDRA procedure described in this paper was shown to be a reliable and rapid method for identifying Lactobacillus species from intestinal and vaginal microflora at species and subspecies level.     

Assessment of microbial community structure changes by amplified ribosomal DNA restriction analysis (ARDRA).

Amplified ribosomal DNA restriction analysis (ARDRA) is a simple method based on restriction endonuclease digestion of the amplified bacterial 16S rDNA. In this study we have evaluated the suitability of this method to detect differences in activated sludge bacterial communities fed on domestic or industrial wastewater, and subject to different operational conditions. The ability of ARDRA to detect these differences has been tested in modified Ludzack-Ettinger (MLE) configurations. Samples from three activated sludge wastewater treatment plants (WWTPs) with the MLE configuration were collected for both oxic and anoxic reactors, and ARDRA patterns using double enzyme digestions AluI+MspI were obtained. A matrix of Dice similarity coefficients was calculated and used to compare these restriction patterns. Differences in the community structure due to influent characteristics and temperature could be observed, but not between the oxic and anoxic reactors of each of the three MLE configurations. Other possible applications of ARDRA for detecting and monitoring changes in activated sludge systems are also discussed.     

Genetic diversity study of Chromobacterium violaceum isolated from Kolli Hills by amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD)

Aim: Chromobacterium are saprophytes that cause highly fatal opportunistic infections. Identification and strain differentiation were performed to identify the strain variability among the environmental samples. We have evaluated the suitability of individual and combined methods to detect the strain variations of the samples collected in different seasons. Methods and Results: Amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD) profiles were obtained using four different restriction enzyme digestions (AluI, HaeIII, MspI and RsaI) and five random primers. A matrix of dice similarity coefficients was calculated and used to compare these restriction patterns. ARDRA showed rapid differentiation of strains based on 16S rDNA, but the combined RAPD and ARDRA gave a more reliable differentiation than when either of them was analysed individually. Conclusion: A high level of genetic diversity was observed, which indicates that the Kolli Hills’C. violaceum isolates would fall into at least three new clusters. Significance and Impact of the Study: Results showed a noteworthy bacterial variation and genetic diversity of C. violaceum in the unexplored, virgin forest area.     

Identification of Weissella species by the genus-specific amplified ribosomal DNA restriction analysis

To investigate the generality of efficient double-strand break repair and damage-induced mutagenesis in hyperthermophilic archaea, we systematically measured the effects of five DNA-damaging agents on Sulfolobus acidocaldarius and compared the results to those obtained for Escherichia coli under corresponding conditions. The observed lethality of gamma-radiation was very similar for S. acidocaldarius and E. coli, arguing against unusually efficient double-strand break repair in S. acidocaldarius. In addition, DNA-strand-breaking agents (gamma-radiation or bleomycin), as well as DNA-cross-linking agents (mechlorethamine, butadiene diepoxide or cisplatin) stimulated forward mutation, reverse mutation, and formation of recombinants via conjugation in Sulfolobus cells. Although two of the five DNA-damaging agents failed to revert the E. coli auxotrophs under these conditions, all five reverted S. acidocaldarius auxotrophs.     

Evaluation of amplified ribosomal DNA restriction analysis as a method for the identification of Botryosphaeria species

The polymerase chain reaction was used to amplify a rDNA fragment containing the internal transcribed spacers (ITS1-5.8S-ITS2) and the D1/D2 variable domains of the 28S rDNA from 10 species of the genus Botryosphaeria (Fungi, Ascomycota). Restriction analysis of the amplicons with frequent-cutting endonucleases (amplified ribosomal DNA restriction analysis) allowed the definition of 12 rDNA haplotypes. Each of the rDNA haplotypes could be unambiguously assigned to a single Botryosphaeria species, thus allowing clear identification of all the species tested. Intraspecific polymorphism was very low and detected only in Botryosphaeria parva and Botryosphaeria dothidea. Cluster analysis of banding patterns of the isolates corresponded well with known species delineations. The method described in this paper provides a simple and rapid procedure for the differentiation and identification of Botryosphaeria isolates at the species level.     

Detection and identification of Lactobacillus species in crops of broilers of different ages by using PCR-denaturing gradient gel electrophoresis and amplified ribosomal DNA restriction analysis

The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 10(8) to 10(9) CFU per gram of crop contents. Many of the lactobacilli present in the crop (61.9% of isolates) belonged to species of the Lactobacillus acidophilus group and could not be differentiated by PCR-DGGE. A rapid and simple ARDRA method was developed to distinguish between the members of the L. acidophilus group. HaeIII-ARDRA was used for preliminary identification of isolates in the L. acidophilus group and to identify Lactobacillus reuteri and Lactobacillus salivarius. MseI-ARDRA generated unique patterns for all species of the L. acidophilus group, identifying Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus gallinarum among crop isolates. The results of our study provide comprehensive knowledge of the Lactobacillus microflora in the crops of birds of different ages using nucleic acid-based methods of detection and identification based on current taxonomic criteria.     

Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of cultured mycobacteria in a diagnostic laboratory

Background

The development of DNA amplification for the direct detection of M. tuberculosis from clinical samples has been a major goal of clinical microbiology during the last ten years. However, the limited sensitivity of most DNA amplification techniques restricts their use to smear positive samples. On the other hand, the development of automated liquid culture has increased the speed and sensitivity of cultivation of mycobacteria. We have opted to combine automated culture with rapid genotypic identification (ARDRA: amplified rDNA restriction analysis) for the detection resp. identification of all mycobacterial species at once, instead of attempting direct PCR based detection from clinical samples of M. tuberculosis only.

Results

During 1998–2000 a total of approx. 3500 clinical samples was screened for the presence of M. tuberculosis. Of the 151 culture positive samples, 61 were M. tuberculosis culture positive. Of the 30 smear positive samples, 26 were M. tuberculosis positive. All but three of these 151 mycobacterial isolates could be identified with ARDRA within on average 36 hours. The three isolates that could not be identified belonged to rare species not yet included in our ARDRA fingerprint library or were isolates with an aberrant pattern.

Conclusions

In our hands, automated culture in combination with ARDRA provides with accurate, practically applicable, wide range identification of mycobacterial species. The existing identification library covers most species, and can be easily updated when new species are studied or described. The drawback is that ARDRA is culture-dependent, since automated culture of M. tuberculosis takes on average 16.7 days (range 6 to 29 days). However, culture is needed after all to assess the antibiotic susceptibility of the strains.

     

Random amplified ribosomal DNA restriction analysis for rapid identification of thermophilic Actinomycete-like bacteria involved in hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is a pulmonary disease characterised by inflammation that can be caused by, amongst other substances, a subset of 4 thermophilic mycelial bacteria: Saccharopolyspora rectivirgula, Saccharomonospora viridis, Thermoactinomyces sacchari, and Thermoactinomyces vulgaris. Air sampling analyses in highly contaminated environments are often performed to evaluate exposure to these species which are difficult and fastidious to identify by conventional techniques. The aim of this study was to use amplified ribosomal DNA restriction analysis (ARDRA) to develop a method of identification for those thermophilic organisms that would be more rapid and simple. Strains of these 4 species were obtained from the American type culture collection (ATCC) and were characterized using biochemical tests and ARDRA patterns obtained on their partial-lenght amplified 16S rDNAs. To validate this approach, ARDRA with two restriction enzymes, TaqI and HhaI, was applied to 49 thermophilic actinomycete-like strains from environmental samples (sawmills). The results obtained show that combining some cultural characteristics and biochemical tests, such as xanthine or hypoxanthine decomposition, growth in the presence of NaCl, lysozyme or novobiocin, and spore resistance over 100 degrees C provide a rough identification and selection of the genera of interest. Consequently, target species could be confirmed by digestion of partial-lenght 16S rDNA with the use of Taql and HhaI restriction enzymes that gave specific restriction patterns. ARDRA analyses on the 49 environmental actinomycete-like organisms revealed the presence of 8 Saccharopolyspora rectivirgula, 2 Saccharomonospora viridis, and 15 Thermoactinomyces vulgaris strains, the other strains had restriction patterns different than those of the species of interest. Results of the present study will be applicable to other potential HP environments such as dairy barns, peat bogs and compost plants.     

Determining potential probiotic properties of human originated Lactobacillus plantarum strains

In this study, twenty Lactobacillus plantarum strains which were isolated from the fecal samples of humans were investigated in vitro for their characteristics as potential new probiotic strains. The L. plantarum strains were examined for resistance to gastric acidity in simulated gastric juice at pH 2.0, 2.5, 3.0, and 3.5. The growth of test cultures with different pH was monitored after 0, 10, 30, 60, 90, and 120 min of incubation using a spectrophotometer at 550 nm. At the same time, samples were serially diluted in sterile PBS, and counts of viable bacteria were determined by plate counts using MRS agar for each pH and time parameter. The strains were also examined for resistance to 0.4% phenol, production of H₂O₂, adhesion to Caco-2 cell line and antimicrobial activity. It was determined that the artificial gastric juice, even at pH 2.0, did not significantly change the viability of the cultures. Except L. plantarum AA1-2, all strains were detected at 8 ～ 9 log₁₀ CFU/g. It was found that all L. plantarum strains showed good resistance to 0.4% phenol, and only one strain (AC18-82) produced H₂O₂. Good adhesion of L. plantarum strains to Caco-2 cells was observed in this experiment. These selected strains also showed antimicrobial activity.     

Genetic diversity of Azotobacter strains isolated from soils by amplified ribosomal DNA restriction analysis

Strains of Azotobacter mediate in the nitrogen fixation process by reducing of N₂ to ammonia. In this study, 50 strains were isolated from different rhizospheric soil in central Iran, by using soil paste-plate method. These strains were biochemically identified and characterized on differential LG medium based on morphological and physiological properties. Results obtained showed that identified strains were belonging to three species, namely A. chroococcum, A. vinelandii and A. beijernckii. In order to molecular analysis, the 16S rRNA gene was amplified using 27f and 1495r primers and PCR products were subsequently restricted with RsaI, HpaII and HhaI. Cluster analysis based on amplified ribosomal DNA restriction analysis were revealed intraspecific polymorphism and differentiated strains into two mains clusters, clusters A and B. Cluster A strains were related to the A. vinelandii, whereas cluster B strains were related to the A. chroococcum and A. beijerinckii. The results show that amplified ribosomal DNA restriction analysis is a powerful and discriminatory tool for the identification of members of the genus Azotobacter.     

Differentiation and identification of iron-oxidizing acidophilic bacteria using cultivation techniques and amplified ribosomal DNA restriction enzyme analysis

Acidophilic iron-oxidizing microorganisms are important both environmentally and in biotechnological applications. Although, as a group, they are readily detected by their ability to generate ferric iron (resulting in a distinctive color change in liquid media), these microbes highly diverse phylogenetically. Various other characteristics, such as optimum growth temperature, response to organic carbon sources, and cellular morphologies, facilitate, in some cases, identification of isolates to a genus or species level, although this approach has limitations and may give erroneous results. In this study, a combined approach of using physiological traits together with amplified ribosomal DNA restriction enzyme analysis (ARDREA) has been successful in identifying all known acidophilic iron-oxidizing bacteria to the species level. Computer-generated maps were used to identify restriction enzymes that allow the differentiation of the acidophiles, and these were confirmed experimentally using authentic bacterial strains. To test further the validity of this approach, six acidophilic moderately thermophilic iron-oxidizing bacteria isolated from Montserrat (West Indies) were analysed using the ARDREA protocol. Three of the isolates were identified as Sulfobacillus acidophilus-like, and one as Sulfobacillus thermosulfidooxidans-like bacteria. The fifth isolate gave DNA digest patterns that were distinct from all known strains of iron-oxidizing acidophiles. Subsequent sequencing of the 16S rRNA genes of these isolates confirmed the identity of the four Sulfobacillus isolates, and also that the fifth isolate was a novel species. Schematic diagrams showing how ARDREA may be used to rapidly identify all known acidophilic iron-oxidizing bacteria are presented.     

Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis

To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.     

Randomly amplified polymorphic DNA (RAPD) profiles for the distinction of Lactobacillus species

Forty-one type and reference strains of Lactobacillus were evaluated using their randomly amplified polymorphic DNA band profiles. Developed bands for each strain were distinct and enabled discrimination. The best correlations were obtained applying the Pearson product moment correlation coefficient (r) together with the unweighted pair group method using arithmetic averages algorithm. All of the strains were clearly differentiated at and below the 72% similarity value. Species discrimination might be possible making use of the distinctly polymorphic bands amplified specific to a strain.     

Evaluation of random amplified polymorphic DNA for species identification and phylogenetic analysis inStylosanthes (Fabaceae)

Random amplified polymorphic DNA (RAPD) was assessed for its suitability as a tool to be used in the identification of taxa from the genusStylosanthes (Fabaceae, Papilionoideae, Aeschynomeneae). Five random primers were used to fingerprint accessions from seven species in the genus, and generated RAPD profiles that were species-specific. Data were used to examine evolutionary relationships between taxa, employing both clustering and ordination techniques, and the results were compared with those from a previous cladistic analysis of chloroplast DNA (cpDNA) restriction fragments. Both multivariate approaches indicated relationships that were generally similar to those obtained by RFLP analysis of cpDNA. However, while cluster analysis grouped together all accessions within species, ordination placed certain accessions ofS. humilis, S. macrocephala andS. capitata into separate groups. Experiments to test the assumed homology of comigrating RAPDs estimated 85.7% homology for accessions within species, and 53.8% homology for accessions between species. The value of RAPD data in systematics is discussed.