Application of a clustering-based peak alignment algorithm to analyze various DNA fingerprinting data

DNA fingerprinting analysis such as amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic PCR (rep-PCR), ribosomal intergenic spacer analysis (RISA), and denaturing gradient gel electrophoresis (DGGE) are frequently used in various fields of microbiology. The major difficulty in DNA fingerprinting data analysis is the alignment of multiple peak sets. We report here an R program for a clustering-based peak alignment algorithm, and its application to analyze various DNA fingerprinting data, such as ARDRA, rep-PCR, RISA, and DGGE data. The results obtained by our clustering algorithm and by BioNumerics software showed high similarity. Since several R packages have been established to statistically analyze various biological data, the distance matrix obtained by our R program can be used for subsequent statistical analyses, some of which were not previously performed but are useful in DNA fingerprinting studies.     

Identification of Bifidobacterium species using rep-PCR fingerprinting

The aim of the present study was to evaluate the use of repetitive DNA element PCR fingerprinting (rep-PCR) for the taxonomic discrimination among the currently described species within the genus Bifidobacterium. After evaluating several primer sets targeting the repetitive DNA elements BOX, ERIC, (GTG)s and REP, the BOXA1R primer was found to be the most optimal choice for the establishment of a taxonomical framework of 80 Bifidobacterium type and reference strains. Subsequently, the BOX-PCR protocol was tested for the identification of 48 unknown bifidobacterial isolates originating from human faecal samples and probiotic products. In conclusion, rep-PCR fingerprinting using the BOXA1R primer can be considered as a promising genotypic tool for the identification of a wide range of bifidobacteria at the species, subspecies and potentially up to the strain level.     

Sample size, library composition, and genotypic diversity among natural populations of Escherichia coli from different animals influence accuracy of determining sources of fecal pollution

A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of Escherichia coli. Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP DNA fingerprints from E. coli strains isolated from fecal material from known human and 12 animal sources: dogs, cats, horses, deer, geese, ducks, chickens, turkeys, cows, pigs, goats, and sheep. HFERP DNA fingerprinting reduced within-gel grouping of DNA fingerprints and improved alignment of DNA fingerprints between gels, relative to that achieved using rep-PCR DNA fingerprinting. Jackknife analysis of the complete rep-PCR DNA fingerprint library, done using Pearson's product-moment correlation coefficient, indicated that animal and human isolates were assigned to the correct source groups with an 82.2% average rate of correct classification. However, when only unique isolates were examined, isolates from a single animal having a unique DNA fingerprint, Jackknife analysis showed that isolates were assigned to the correct source groups with a 60.5% average rate of correct classification. The percentages of correctly classified isolates were about 15 and 17% greater for rep-PCR and HFERP, respectively, when analyses were done using the curve-based Pearson's product-moment correlation coefficient, rather than the band-based Jaccard algorithm. Rarefaction analysis indicated that, despite the relatively large size of the known-source database, genetic diversity in E. coli was very great and is most likely accounting for our inability to correctly classify many environmental E. coli isolates. Our data indicate that removal of duplicate genotypes within DNA fingerprint libraries, increased database size, proper methods of statistical analysis, and correct alignment of band data within and between gels improve the accuracy of microbial source tracking methods.     

Application of PCR, rep-PCR and RAPD techniques for typing ofLactococcus lactis strains

A collection of 34 lactococcal strains were characterized using the polymerase chain reaction (PCR) for the acmA gene, and for the 16S rDNA gene, and DNA fingerprinting methods for randomly amplified polymorphic DNA (RAPD) and repetitive extragenic palindrome-PCR (rep-PCR). PCR experiments corroborated the genotypic identification of Lactococcus lactis strains by RAPD; rep-PCR did not distinguish between L. lactis subspecies. In some cases, phenotypic classification of L. lactis subspecies did not correlate with genotypic characterization.     

A comparative evaluation of five typing techniques for determining the diversity of fluorescent pseudomonads

Five typing methods were evaluated, utilising 63 strains of fluorescent pseudomonads, to assess their usefulness as tools to study the bacterial diversity within this complex group. The methods used were Biolog metabolic profiling, restriction fragment length polymorphism ribotyping, PCR ribotyping, and repetitive element sequence-based PCR (rep-PCR) utilising BOX and enterobacterial repetitive intergenic consensus (ERIC) primers. Cluster analysis of the results clearly demonstrated the considerable homogeneity of Pseudomonas aeruginosa isolates and, conversely, the heterogeneity within the other species, in particular P. putida and P. fluorescens, which need further taxonomic investigation. Biolog metabolic profiling enabled the best differentiation among the species. Rep-PCR proved to be highly discriminatory, more so than the other DNA fingerprinting techniques, demonstrating its suitability for the analysis of highly clonal isolates. RFLP ribotyping, PCR ribotyping, and rep-PCR produced specific clusters of P. aeruginosa isolates, which corresponded to their origins of isolation, hence we recommend these methods for intraspecific typing of bacteria.     

Strain-specific fingerprints of Rhizobium galegae generated by PCR with arbitrary and repetitive primers

Strain-specific genomic patterns of Rhizobium galegae were generated by PCR using both arbitrary and repetitive (BOX, ERIC and REP) primers. The identification of the strains was achieved also by RFLP analysis. However, the PCR genomic fingerprinting has significant advantages: it is not only simpler and faster, but it is also much more discriminative because it deals with the full bacterial genome and not only with parts of it as is the case with RFLP. In addition, both kinds of PCR fingerprinting (using arbitrary or repetitive primers) generated highly specific and reproducible patterns when parallel reactions with total bacterial DNA, extracted from independent liquid cultures were performed. The latter shows that AP- and rep-PCR are convenient for controlling the production and application of Rhizobium inoculants.     

Diversity of DNA sequences among Vibrio cholerae O1 and non-O1 isolates detected by whole-cell repetitive element sequence-based polymerase chain reaction

Vibrio cholerae strains isolated from patient, food and environmental sources in Taiwan and reference V. cholerae strains were examined by repetitive element sequence-based PCR (rep-PCR). Specimens from broth cultures were used directly in the PCR mixture with three different primers. The PCR fingerprinting profiles of toxigenic 01 isolates were not only homogeneous with primers from enterobacterial repetitive intergenic consensus (ERIC) sequences, but also allowed the differentiation from non-toxigenic O1 and non-O1 strains. Toxigenic 01 strains were further differentiated into El Tor and classical biotypes with primers designed from ERIC-related sequences of V. cholerae. Primers from the other V. cholerae repetitive DNA sequences, VCR, separated toxigenic El Tor strains into six groups and a unique pattern was also obtained in 16 isolates from imported cases of cholera and imported seafood. The results indicated that rep-PCR can be used to identify and differentiate different toxigenic 01, non-toxigenic 01 and non-O1 V. cholerae isolates.     

Rep-PCR - a variant to RAPD or an independent technique of bacteria genotyping? A comparison of the typing properties of rep-PCR with other recognised methods of genotyping of microorganisms

The paper presents technical aspects of rep-PCR fingerprinting technique and compares its typing abilities, differentiation power and reproducibility with other recognised and recommended genotyping methods. Although rep-PCR fingerprinting is similar to MAAP techniques, it demonstrates some essentially different elements. The data presented in this review, indicate a rep-PCR genomic fingerprinting technique as a highly discriminating, independent screening method for determining the taxonomical diversity of bacterial population.     

BOX-PCR is an Adequate Tool for Typing <Emphasis Type="Italic">Aeromonas</Emphasis> spp.

PCR-based methods of fingerprinting take advantage of the presence of repetitive sequences that are interspersed throughout the genome of diverse bacterial species. They include the repetitive extragenic palindromic (REP) sequence, the enterobacterial repetitive intergenic consensus sequence (ERIC) and the 154-bp BOX element. The combination of the three methods is used for fine discrimination of strains and is designated as rep-polymerase chain reaction (PCR). REP-PCR and ERIC-PCR have been shown to be useful for typing Aeromonas strains. To our knowledge, rep-PCR fingerprinting method using the BOXA1R primer has never been tested on aeromonads. In this study, the BOX-PCR fingerprinting technique was evaluated for the discrimination of strains of some Aeromonas species. All strains were typeable and the majority showed unique banding patterns. Four strains from culture collections were used to investigate the reproducibility of the method. According to our results, BOX-PCR fingerprinting is applicable for typing of Aeromonas strains and can be considered as a useful complementary tool for epidemiological studies of members of this genus.     

Bacteriocin Typing of Burkholderia (Pseudomonas) solanacearum Race 1 of the French West Indies and Correlation with Genomic Variation of the Pathogen

Burkholderia solanacearum race 1 isolates indigenous to the French West Indies were characterized by bacteriocin typing and two genomic fingerprinting methods: pulsed-field gel electrophoresis of genomic DNA digested by rare-cutting restriction endonucleases (RC-PFGE) and PCR with primers corresponding to repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and BOX elements (collectively known as rep-PCR). The survey comprised 24 reference strains and 65 isolates obtained from a field trial in Guadeloupe in 1993. Comparison of the data identified RC-PFGE as the most discriminatory method, delineating 17 pulsed-field gel profile types. rep-PCR and bacteriocin typing identified nine rep-PCR profile types and nine bacteriocin groups. Independent determination of similarity coefficients and clustering of RC-PFGE and rep-PCR data identified six groups common to both sets of data that correlated to biovar and bacteriocin groups. Further study of bacteriocin production in planta gave results consistent with in vitro bacteriocin typing. It was observed that spontaneous bacteriocin-resistant mutants exhibited a cross-resistance to other bacteriocins as identified by the typing scheme and that such mutants possessed a selective advantage for growth over isogenic nonmutants in the presence of a bacteriocin. The results are significant in the search for biological control of disease by nonpathogenic mutants of the wild-type organism.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

Rep-PCR mediated genomic fingerprinting of rhizobia and computer-assisted phylogenetic pattern analysis

A rapid and reproducible method has been developed for genomic fingerprinting of rhizobia and other soil microbes interacting with plants. The method is based on the use of oligonucleotide primers, corresponding to conserved motifs in naturally occurring interspersed repetitive DNA elements in bacteria (rep-elements), and the polymerase chain reaction (rep-PCR). Rep-PCR results in the amplification of inter-element genomic DNA fragments of characteristic lengths and thereby generates a genomic fingerprint. These fingerprints resemble UPC bar code patterns, and can be used to identify bacteria at the sub-species and strain level, as well as for phylogenetic analyses. Here we show that highly characteristic and very reproducible rep-PCR generated genomic fingerprints can be obtained not only from purified genomic DNA, but also directly from rhizobial cells derived from liquid cultures or from colonies on plates, as well as from nodule tissue. We examine the effect of growth phase of the bacterial cells, serial subculturing and other parameters on the reproducibility of the rep-PCR fingerprinting protocol. Moreover, we describe the results of mixing experiments designed to determine if individual genomic fingerprints can be recognized in mixtures of strains. Lastly, we review the use of computer-based fragment detection and phylogentic analysis packages to analyse rep-PCR generated genomic fingerprints of a collection of Rhizobium loti and Bradyrhizobium strains nodulating different Lotus spp.The authors are with the NSF Center for Microbial Ecology and the MSU-DOE Plant Research Laboratory. F. J. de Bruijn is also with the Microbiology Department and Genetics Program of Michigan State University, E. Lansing, MI 48824 USA.     

Evaluation of repetitive extragenic palindromic-PCR for discrimination of fecal Escherichia coli from humans, and different domestic- and wild-animals

The objective of this study was to investigate the potential of repetitive extragenic palindromic anchored polymerase chain reaction (rep-PCR) in differentiating fecal Escherichia coli isolates of human, domestic- and wild-animal origin that might be used as a molecular tool to identify the possible source(s) of fecal pollution of source water. A total of 625 fecal E. coli isolates of human, 3 domestic- (cow, dog and horse) and 7 wild-animal (black bear, coyote, elk, marmot, mule deer, raccoon and wolf) species were characterized by rep-PCR DNA fingerprinting technique coupled with BOX A1R primer and discriminant analysis. Discriminant analysis of rep-PCR DNA fingerprints of fecal E. coli isolates from 11 host sources revealed an average rate of correct classification of 79.89%, and 84.6%, 83.8%, 83.3%, 82.5%, 81.6%, 80.8%, 79.8%, 79.3%, 77.4%, 73.2% and 63.6% of elk, human, marmot, mule deer, cow, coyote, raccoon, horse, dog, wolf and black bear fecal E. coli isolates were assigned to the correct host source. These results suggest that rep-PCR DNA fingerprinting procedures can be used as a source tracking tool for detection of human- as well as animal-derived fecal contamination of water.     

A novel means to develop strain-specific DNA probes for detecting bacteria in the environment.

A simple means to develop strain-specific DNA probes for use in monitoring the movement and survival of bacteria in natural and laboratory ecosystems was developed. The method employed amplification of genomic DNA via repetitive sequence-based PCR (rep-PCR) using primers specific for repetitive extragenic palindromic (REP) elements, followed by cloning of the amplified fragments. The cloned fragments were screened to identify those which were strain specific, and these were used as probes for total genomic DNA isolated from microbial communities and subjected to rep-PCR. To evaluate the utility of the approach, we developed probes specific for Burkholderia cepacia G4 and used them to determine the persistence of the strain in aquifer sediment microcosms following bioaugmentation. Two of four probes tested were found to specifically hybridize to DNA fragments of the expected sizes in the rep-PCR fingerprint of B. cepacia G4 but not to 64 genetically distinct bacteria previously isolated from the aquifer. One of these probes, a 650-bp fragment, produced a hybridization signal when as few as 10 CFU of B. cepacia G4 were present in a mixture with 10(6) CFU nontarget strains, indicating that the sensitivity of these probes was comparable to those of other PCR-based detection methods. The probes were used to discriminate groundwater and microcosm samples that contained B. cepacia G4 from those which did not. False-positive results were obtained with a few samples, but these were readily identified by using hybridization to the second probe as a confirmation step. The general applicability of the method was demonstrated by constructing probes specific to three other environmental isolates.     

Isolation of endophytic bacteria from rice and assessment of their potential for supplying rice with biologically fixed nitrogen

The extension of nitrogen-fixing symbioses to important crop plants such as the cereals has been a long-standing goal in the field of biological nitrogen fixation. One of the approaches that has been used to try to achieve this goal involves the isolation and characterization of stable endophytic bacteria from a variety of wild and cultivated rice species that either have a natural ability to fix nitrogen or can be engineered to do so. Here we present the results of our first screening effort for rice endophytes and their characterization using acetylene reduction assays (ARA), genomic fingerprinting with primers corresponding to naturally occurring repetitive DNA elements (rep-PCR), partial 16S rDNA sequence analysis and PCR mediated detection of nitrogen fixation (nif) genes with universal nif primers developed in our laboratory. We also describe our efforts to inoculate rice plants with the isolates obtained from the screening, in order to examine their invasiveness and persistence (stable endophytic maintenance). Lastly, we review our attempts to tag selected isolates with reporter genes/proteins, such as beta-glucuronidase (gus) or green fluorescent protein (gfp), in order to be able to track putative endophytes during colonization of rice tissues.     

Characterization of Rhizobium 'hedysari' by RFLP analysis of PCR amplified rDNA and by genomic PCR fingerprinting

The taxonomic and discriminatory power of RFLP analysis of PCR amplified parts of rhizobial rrn operons was compared to those of genomic PCR fingerprinting with arbitrary and repetitive primers. For this purpose, the two methods were applied for characterization of a group of bacterial isolates referred to as Rhizobium 'hedysari'. As outgroups, representatives of the family Rhizobiaceae, belonging to the Rhizobium galegae, Rhizobium meliloti, Rhizobium leguminosarum and Agrobacterium tumefaciens species were used. By the RFLP analysis of the PCR products corresponding to the variable 5'-half of the 23S rRNA gene and of the amplified spacer region between the 16S and 23S rRNA genes all Rh. 'hedysari' strains studied were tightly clustered together while the outgroups were placed in an outer position. The PCR products of the 3' end parts of the 23S rDNA did not show significant RFL polymorphism and no species differentiation on their basis was possible. In parallel, analysis of the same strains was performed by PCR amplification of their total DNA with 19, 18 and 10 bp long arbitrary primers (AP-PCR) as well as with single primers corresponding to several bacterial repetitive sequences (rep-PCR). By both AP and rep-PCR an identification of every particular strain was achieved. In general, all primers provided taxonomic results that are in agreement with the species and group assignments based on the RFLP analysis of the rrn operons. On the basis of the results presented here it can be concluded that AP and rcp-PCR are more informative and discriminative than rDNA and RFLP analysis of the rhizobial strains studied.     

Use of the DiversiLab repetitive sequence-based PCR system for genotyping and identification of Archaea

Repetitive elements are short stretches of DNA that are randomly distributed throughout the chromosomes of prokaryotes. The use of PCR primers to amplify intervening sequences of DNA between specific repetitive elements in Bacteria has become a standard method for rapidly genotyping bacterial strains and providing good resolution between multiple strains within a single species. Rapid, standardized methods for high resolution genotyping of Archaea are not widely available. We evaluated the DiversiLab system from Bacterial Barcodes that utilizes a kit-based repetitive sequence-based (rep-PCR) method that has been optimized for genotyping DNA was extracted from the source organisms using either a standard chemical DNA extraction kit or Whatman FTA paper. Rep-PCR was performed using an archaeal primer set and, the products were run on an Agilent, Lab-on-a-Chip DNA analyzer. Results were analyzed and compared using DiversiLab web-based software from Bacterial Barcodes. Seventy-nine strains representing 27 genera of Crenarchaeota and Euryarchaeota were analyzed. All the organisms could be successfully genotyped and the results were reproducible. We could not detect differences in rep-PCR profiles between DNA extracted using the chemical extraction kit and FTA paper. Thus far, 14 genera and 32 species of methanogens have been analyzed, and all yielded unique genotypes. For halophiles, 11 genera and 28 different species were analyzed, and all yielded unique genotypes. A comparison of 7 different strains of Halobacterium salinarium demonstrated that 6 of the 7 strains had a unique genotype. A comparison of 4 strains of Methanosarcina mazei indicated that each strain produced a unique genotype. There was little systematic inference that could be made from dendrograms comparing different strains, species, and genera of Archaea based on UPGMA cluster analysis. Based on these results, rep-PCR was a useful tool for the genotyping and strain identification of Archaea.     

Genomic fingerprints of Salmonella species generated with repetitive element sequence-based PCR

We describe the use of repetitive element sequence-based PCR (rep-PCR) on the two repetitive sequences, REP and ERIC elements, to distinguish members of closely related Salmonella species. Within the species, ERIC–PCR showed a higher discriminative potential than REP–PCR, but by using a combination of the two PCR methods it was possible to distinguish all the isolates examined. The rep-PCR fingerprints of Salmonella organisms were distinctly different from some Gram-positive bacteria, for example Staphylococcus, Bacillus megaterium, and even the closely related Escherichia coli and Serratia marcescens. Identical fingerprints were observed with whole-cell preparations. Rapid specimen preparation has enhanced the value of rep-PCR in timely analysis of epidemiological relationships.     

In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities.

Obtaining information on the genetic capabilities and phylogenetic affinities of individual prokaryotic cells within natural communities is a high priority in the fields of microbial ecology, microbial biogeochemistry, and applied microbiology, among others. A method for prokaryotic in situ PCR (PI-PCR), a technique which will allow single cells within complex mixtures to be identified and characterized genetically, is presented here. The method involves amplification of specific nuclei acid sequences inside intact prokaryotic cells followed by color or fluorescence detection of the localized PCR product via bright-field or epifluorescence microscopy. Prokaryotic DNA and mRNA were both used successfully as targets for PI-PCR. We demonstrate the use of PI-PCR to identify nahA-positive cells in mixtures of bacterial isolates and in model marine bacterial communities.     

Identification and characterization of thermophilic bacteria isolated from hot springs in Turkey

The present study was conducted to identify and characterize the thermophilic bacteria isolated from various hot springs in Turkey by using phenotypic and genotypic methods including fatty acid methyl ester and rep-PCR profilings, and 16S rRNA sequencing. The data of fatty acid analysis showed the presence of 17 different fatty acids in 15 bacterial strains examined in this study. Six fatty acids, 15:0 iso, 15:0 anteiso, 16:0, 16:0 iso, 17:0 iso, and 17:0 anteiso, were present in all strains. The bacterial strains were classified into three phenotypic groups based on fatty acid profiles which were confirmed by genotypic methods such as 16S rRNA sequence analysis and rep-PCR genomic fingerprint profiles. After evaluating several primer sets targeting the repetitive DNA elements of REP, ERIC, BOX and (GTG)₅, the (GTG)₅ and BOXA1R primers were found to be the most reliable technique for identification and taxonomic characterization of thermophilic bacteria in the genera of Geobacillus, Anoxybacillus and Bacillus spp. Therefore, rep-PCR fingerprinting using the (GTG)₅ and BOXA1R primers can be considered as a promising genotypic tool for the identification and characterization of thermophilic bacteria from species to strain level.