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.     

Genetic diversity of Bradyrhizobium japonicum field population revealed by RAPD fingerprinting

RAPD fingerprinting was used for strain identification and the assessment of genetic diversity within a field population of Bradyrhizobium japonicum . Total genomic DNAs from 13 field isolates and two inoculant strains were amplified using six different 10-mer primers. Different and informative band patterns were obtained for all strains analysed. Cluster analysis unexpectedly revealed that none of the field isolates was identical to inoculant strains which were regularly used for soybean inoculation. Among field isolates two highly divergent groups were determined. The results indicate that RAPD is a very discriminative and efficient method for differentiating and studying genetic diversity of B. japonicum strains.     

RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains.

The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.     

Repetitive elements sequence (REP/ERIC)-PCR based genotyping of clinical and environmental strains of Yersinia enterocolitica biotype 1A reveal existence of limited number of clonal groups

REP- and ERIC-PCR genotyping were used to assess genetic heterogeneity among 81 strains of Yersinia enterocolitica biotype 1A isolated from India, Germany, France and the USA. Although both gave comparable results, ERIC fingerprints discriminated the strains better. The rep- (REP and ERIC) PCR genotyping showed that strains having different serotypes produced identical rep-profiles indicating their limited genetic diversity. The concatenated dendrogram of REP- and ERIC-PCR fingerprints clustered the biotype 1A strains into two major groups. In each group, majority of the Indian, European and American strains exhibited similarities ranging from 85% to >95%. Similarity of rep-PCR fingerprints amongst strains isolated from widely separated geographical regions revealed existence of a limited number of clonal groups of Y. enterocolitica biotype 1A. The present study failed to reveal unequivocal relationships between rep-PCR genotypes and the source of isolation. However, the clinical serotype O:6,30-6,31 strains formed a tight cluster and the aquatic O:6,30-6,31 strains formed a yet another tight cluster.     

RAPD and rep-PCR fingerprinting for characterization of <Emphasis Type="Italic">Bifidobacterium</Emphasis> species

DNA fingerprinting methods, RAPD with 7 random primers, and rep-PCR using both BOXA1R and (GTG)(5) ones, were used for the discrimination of 16 type and collection Bifidobacterium strains of 9 species of human origin, B. animalis ssp. animalis and B. animalis ssp. lactis and 7 Bifidobacterium strains collected in the Culture Collection of Dairy Microorganisms (CCDM). Both RAPD and rep-PCR methods provided similar results. The strains were identified as B. animalis ssp. lactis (6 strains) and B. adolescentis (1 strain). The reclassification of the collection strain CCM 3761 as B. pseudocatenulatum species (previously classified as B. adolescentis) was confirmed.     

Effectiveness of enterobacterial repetitive intergenic consensus PCR and random amplified polymorphic DNA fingerprinting for Helicobacter pylori strain differentiation

We compared the robustness and discriminatory power of the enterobacterial repetitive intergenic consensus (ERIC) and random amplified polymorphic DNA (RAPD) fingerprinting methods for detecting cases of mixed Helicobacter pylori infection in Peruvian shantytown residents. H. pylori isolates from 63 participants were cultured, and five single colonies and a pool of additional colonies from each participant were analyzed by ERIC-PCR and by RAPD tests with four 10-nucleotide primers (one primer per reaction). There was 94% agreement between the ERIC and RAPD profiles in classifying sets of isolates as uniform versus closely related but not identical versus probably unrelated, indicating a high kappa statistic of 0.8942. Subtle differences in related ERIC or RAPD patterns likely reflect gene transfer between strains, recombination, and/or mutation, whereas markedly different patterns reflect infection by unrelated strains. At least half of infected shantytown residents seemed to carry more than one H. pylori strain, although in 19 of 31 persons, the strains were closely related. Three RAPD tests, each with a different primer, were needed to achieve the sensitivity of one ERIC test. ERIC-PCR constitutes a resource- and time-efficient method for H. pylori strain differentiation.     

Analysis of genomic diversity among photosynthetic stem-nodulating rhizobial strains from northeast Argentina

The genomic diversity among photosynthetic rhizobia from northeast Argentina was assessed. Forty six isolates obtained from naturally occurring stem and root nodules of Aeschynomene rudis plants were analyzed by three molecular typing methods with different levels of taxonomic resolution: repetitive sequence-based PCR (rep-PCR) genomic fingerprinting with BOX and REP primers, amplified 16S rDNA restriction analysis (ARDRA), and 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (IGS-RFLP) analysis. The in vivo absorption spectra of membranes of strains were similar in the near infrared region with peaks at 870 and 800 nm revealing the presence of light harvesting complex I, bacteriochlorophyll-binding polypeptides (LHI-Bchl complex). After extraction with acetone-methanol the spectra differed in the visible part displaying peaks belonging to canthaxanthin or spirilloxanthin as the main carotenoid complement. The genotypic characterization by rep-PCR revealed a high level of genomic diversity among the isolates and almost all the photosynthetic ones have identical ARDRA patterns and fell into one cluster different from Bradyrhizobium japonicum and Bradyrhizobium elkanii. In the combined analysis of ARDRA and rep-PCR fingerprints, 7 clusters were found including most of the isolates. Five of those contained only photosynthetic isolates; all canthaxanthin-containing strains grouped in one cluster, most of the other photosynthetic isolates were grouped in a second large cluster, while the remaining three clusters contained a few strains. The other two clusters comprising reference strains of B. japonicum and B. elkanii, respectively. The IGS-RFLP analysis produced similar clustering for almost all the strains. The 16S rRNA gene sequence of one representative isolate was determined and the DNA sequence analysis confirmed the position of photosynthetic rhizobia in a distinct phylogenetic group within the Bradyrhizobium rDNA cluster.     

Intraspecific genetic variability of Edwardsiella tarda strains from cultured turbot

Edwardsiella tarda is an enterobacterial fish pathogen that causes mortality in various fish species worldwide. In this study, we analyzed the intraspecific variability in a collection of E. tarda strains isolated from turbot. To do this we employed 4 polymerase chain reaction (PCR)-based methods: (1) random amplified polymorphic DNA (RAPD), (2) enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), (3) repetitive extragenic palindromic-PCR (REP-PCR) and (4) BOX-PCR. E. tarda isolates from different hosts were also included for comparison. E. tarda strains from turbot showed high molecular homogeneity when RAPD (primers P3 and P6), ERIC-PCR and BOX-PCR were employed. However, with regard to the REP-PCR and RAPD (primers P4 and P5) techniques, different genetic groups could be established within these isolates using either technique. The 2 RAPD types presented an 85% similarity, while those obtained with REP-PCR showed 74% similarity. Based on the results obtained, although a high genetic homogeneity was found in turbot isolates, the RAPD test (with primers P4 and P5) and REP-PCR were capable of discrimination within these strains, and they are therefore considered the most appropriate typing methods for studies of edwardsiellosis in turbot.     

Use of RAPD-PCR fingerprinting to detect genetic diversity of soil Streptomyces isolates

Random amplified polymorphic DNA (RAPD) was used for identification and assessment of genetic diversity between isolates of Streptomyces from soil. Genomic DNA from 18 Streptomyces isolates and 2 reference strains were amplified using four different 10-mer primers. Different DNA fingerprinting patterns were obtained for all the isolates. Electrophoretic and cluster analysis of the amplification products revealed incidence of polymorphism among the isolates and none of them was identical to the reference strains although there were some common amplification bands. Two highly divergent groups were determined among the isolates. The results indicate that RAPD is an efficient method for discriminating and studying genetic diversity of Streptomyces isolates.     

Molecular characterization of Bacillus anthracis using multiplex PCR, ERIC-PCR and RAPD

AIMS: To investigate the molecular characterization of Bacillus anthracis strains by multiplex PCR, enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and random amplification of polymorphic DNA (RAPD). METHODS AND RESULTS: Three primers were used to amplify the cya, cap and cereolysinAB genes in the multiplex PCR. Two distinct ERIC-PCR and RAPD fragments, which separated B. anthracis into two groups, were used as probes in Southern hybridization experiments. The probes hybridized only to the cya+ B. anthracis strains identified by the multiplex PCR. Nucleotide sequence analysis of the two cloned fragments showed they were from the pXO1 plasmid of B. anthracis. CONCLUSION: Multiplex PCR simultaneously identified isolates of the Bacillus cereus group and the B. anthracis virulence factors. ERIC-PCR and RAPD, combined with the Southern hybridization analyses, differentiated B. anthracis strains and separated them from the closely related B. cereus group bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: ERIC-PCR and RAPD assay could be effective in differentiating virulent from avirulent B. anthracis. Our results also show that the amplification of the large plasmids was allowed in the ERIC-PCR and RAPD assay.     

Analysis of Salmonella Agona and Salmonella Weltevreden in Malaysia by PCR fingerprinting and antibiotic resistance profiling

Forty-eight strains of Salmonella enterica subsp. enterica serovar Agona and 33 strains of Salmonella enterica subsp. enterica serovar Weltevreden were characterized by random amplified polymorphic DNA (RAPD) fingerprinting using 3 different arbitrary primer, Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) and antimicrobial susceptibility testing. By using RAPD, 81 strains (44 strains of S. Agona and 33 strains of S. Weltevreden) can be clustered into 14 groups and 6 single isolates whereas ERIC-PCR produced 7 clusters and 3 single isolates. Thirteen antimicrobial agents were used and all the isolates were resistant to erythromycin and showed Multiple Antimicrobial Resistance indexes, ranging from 0.08 to 0.62. Poultry still remain as the common reservoir for multi-drug-resistant Salmonella. On the other hand, vegetables contaminated with S. Weltevreden showed a gain in antimicrobial resistance. Besides that, consistent antibiograms were observed from S. Weltevreden isolated at Kajang wet market on 2000/08/02.     

Differentiation of Fecal <Emphasis Type="Italic">Escherichia coli</Emphasis> from Human,Livestock, and Poultry Sources by rep-PCR DNA Fingerprinting on the Shellfish Culture Area of East China Sea

The rep-PCR DNA fingerprinting performed with REP, BOX A1R, and (GTG)₅ primers was investigated as a way to differentiate between human, livestock, and poultry sources of fecal pollution on the area of Xiangshan Bay, East China Sea. Of the three methods, the BOX-PCR DNA fingerprints analyzed by jack-knife algorithm were revealed high rate of correct classification (RCC) with 91.30, 80.39, 89.39, 86.14, 93.24, 87.72, and 89.28% of human, cattle, swine, chicken, duck, sheep, and goose E. coli isolates classified into the correct host source, respectively. The average rate of correct classification (ARCC) of REP-, BOX-, and (GTG)₅-PCR patterns was 79.88, 88.21, and 86.39%, respectively. Although the highest amount of bands in (GTG)₅-PCR fingerprints could be observed, the discriminatory efficacy of BOX-PCR was superior to both REP- and (GTG)₅-PCR. Moreover, the similarity of 459 isolates originated from shellfish and growing water was compared with fecal-obtained strains. The results showed that 92.4 and 96.2% E. coli strains isolated from midstream and downstream shellfish samples, respectively, had a ≥80% similarity with corresponding strains isolated from fecal samples. It was indicated that E. coli in feces could spread from human sewage or domestic farms to the surrounding shellfish culture water, and potentially affect the quality of shellfish. This work suggests that rep-PCR fingerprinting can be a promising genotypic tool applied in the shellfish growing water management on East China Sea for source identification of fecal pollution.     

Random and repetitive primer amplified polymorphic DNA analysis of Bacillus sphaericus

A comparative study of 15 strains representing the five homology groups of Bacillus sphaericus was performed by two PCR methods: RAPD and rep-PCR fingerprinting. The PCR analysis performed with primers corresponding to the naturally occurring repetitive sequences REP, ERIC and BOX, as well as with three random primers, showed highly variable patterns and allowed differentiation of the strains studied. This demonstrated the high discriminative power of the methods. The cluster analysis revealed a low level of similarity between the different homology groups, and within groups I and III, which is evidence of the high genetic heterogeneity of the species B. sphaericus. Close genetic relatedness was observed for the representatives of group IIA, pathogenic to mosquitoes, which supports the idea for differentiation of this group as a separate species.     

Evaluation of selected Listeria monocytogenes genotyping methods

Listeria monocytogenes infections are found in Poland and all world as sporadic episodes and large outbreaks of human illness as well, where bacteria-contaminated food is the source of infection. Genotyping of strains isolated from outbreak is one of many other stages of epidemiologic investigation. In case of L. monocytogenes pulse-field gel electrophoresis (PFGE) - "golden standard" of genotyping and also ERIC-PCR and rep-PCR could be used. The goal of this study was to evaluate the usefulness of methods: PFGE, ERIC-PCR and rep-PCR to Listeria monocytogenes strains genotyping. Moreover estimation of L. monocytogenes strains isolated in Poland during 2003 - 2006 years genetic diversity was performed. In performed studies, 44 strains of L. monocytogenes from PZH strains collection from 2003 - 2006 period were used. Standard strains of L. monocytogenes, L. ivanovii, L. welschimeri and L. innocua were used also. All those strains were genotyped using following methods: PFGE with ApaI and AscI restrictases and random amplification of polymorphic DNA fragments (ERIC-PCR, rep-PCR). Obtained results were analyzed using Tenover method and specialistic software - Gene Profiler (Scan Analytics, USA). In every analysis, clonal groups were obtained - using computer analysis as well as Tenover method. For PFGE with ApaI and AscI six clonal groups were obtained in each analysis. For ERIC-PCR and rep-PCR four clonal groups were obtained (every group was different except one, where four the same strains were included). Taking into account such factors, as efficiency of strains differentiation, work consumption and costs of procedure performing, for quick application, especially in local laboratories, the best genotyping technique is rep-PCR and ERIC-PCR a little less. On account of appreciable costs of PFGE analysis, this method should be used as reference technique.     

Fingerprinting of Mixed Bacterial Strains and BIOLOG Gram-Negative (GN) Substrate Communities by Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR)

PCR-based genomic fingerprinting by use of enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was evaluated for its use in fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG Gram-negative (GN) microplate substrate communities. ERIC-PCR fingerprints of six different pure bacterial strains and a combined mixture of the strains were compared with fingerprints obtained by two more established methods: amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA analysis (RAPD-PCR). The ERIC-PCR fingerprint of the mixed strains was highly reproducible and was more species-specific and representative of the individual strain fingerprints than the ARDRA and RAPD-PCR fingerprints, respectively. ERIC-PCR fingerprinting of model and rhizosphere BIOLOG GN substrate communities also provided clearly distinguishable fingerprints. Results of this study suggest that ERIC-PCR represents a rapid and highly discriminating method for fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG GN substrate communities. Received: 11 September 1998 / Accepted: 29 October 1998     

Genetic diversity of bradyrhizobial populations from diverse geographic origins that nodulate Lupinus spp. and Ornithopus spp

The genetic diversity of 45 bradyrhizobial isolates that nodulate several Lupinus and Ornithopus species in different geographic locations was investigated by 16S rDNA PCR-RFLP and sequence analysis, 16S-23S rDNA intergenic spacer (IGS) PCR-RFLP analysis, and ERIC-PCR genomic fingerprinting. Reference strains of Bradyrhizobium japonicum, B. liaoningense and B. elkanii and some Canarian isolates from endemic woody legumes in the tribe Genisteae were also included. The 16S rDNA-RFLP analysis resolved 9 genotypes of lupin isolates, a group of fourteen isolates presented restriction-genotypes identical or very similar to B. japonicum, while another two main groups of isolates (69%) presented genotypes that clearly separated them from the reference species of soybean. 16S rDNA sequencing of representative strains largely agreed with restriction analysis, except for a group of six isolates, and showed that all the lupin isolates are relatives of B. japonicum, but different lineages were observed. The 16S-23S IGS-RFLP analysis showed a high resolution level, resolving 19 distinct genotypes among 30 strains analysed, and so demonstrating the heterogeneity of the 16S-RFLP groups. ERIC-PCR fingerprint analysis showed an enormous genetic diversity producing a different pattern for each but two of the isolates. Phylogeny of nodC gene was independent from the 16S rRNA phylogeny, and showed a tight relationship in the symbiotic region of the lupin isolates with isolates from Canarian genistoid woody legumes, and in concordance, cross-nodulation was found. We conclude that Lupinus is a promiscuous host legume that is nodulated by rhizobia with very different chromosomal genotypes, which could even belong to several species of Bradyrhizobium. No correlation among genomic background, original host plant and geographic location was found, so, different chromosomal genotypes could be detected at a single site and in a same plant species, on the contrary, an identical genotype was detected in very different geographical locations and plants.     

Application of Random Amplified Polymorphic DNA analysis to differentiate strains of Salmonella typhi and other Salmonella species

A Random Amplified Polymorphic DNA (RAPD) fingerprinting method was developed to differentiate isolates of Salmonella serotype typhi ( S. typhi ) and other Salmonella isolates. A panel of five primers was used to examine 63 isolates of Salm. typhi , including 56 strains isolated in Taiwan and seven strains obtained abroad. Twenty-one RAPD types were revealed using the RAPD fingerprinting method. An RAPD with primer 6032 yielded a polymorphism in a 350 bp fragment that differentiated the attenuated vaccine strain Salm. typhi Ty21a from the rest of the Salm. typhi strains. Strains of Salm. typhi were divided into five types with primer D14307. Primer D14307 also proved capable of discrimination among 65 other Salmonella isolates representing 42 different serotypes. The bacterial DNA used in this RAPD protocol was obtained using a commercially available DNA extraction kit (GeneReleaser). The DNA of various strains of Salmonella from this simple extraction procedure could be discriminated within a few hours using the RAPD technique.     

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.     

Molecular typing of native Bacillus thuringiensis isolates from diverse habitats in India using REP-PCR and ERIC-PCR analysis

Bacillus thuringiensis is a bacterium of great agronomic and scientific interest. The subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specificity. Therefore molecular typing and diversity analysis of B. thuringiensis has enormous importance for discrimination of strains isolated from different sources. In this study, 113 native B. thuringiensis isolates collected from diverse habitats and locations in India and 27 B. thuringiensis type strains obtained from the Bacillus Genetic Stock Centre (BGSC), Ohio State University, USA and used as reference, were analyzed for molecular typing. Genotypic data of 140 B. thuringiensis isolates and type strains was generated by using REP-PCR and ERIC-PCR primers and unweighted pair group method with arithmetic mean (UPGMA) analysis using NTSYSpc2.2 and grouped into 4 main clusters. All the groups have isolates from diverse origins. No group was found to represent any specific origin or location. The observed patterns of REP-PCR and ERIC-PCR pattern were discriminatory enough to reveal differences in the B. thuringiensis isolates and reference strains. The resolution power and marker index of the ERIC-PCR (RP 9.39, MI 6.34) was found to be higher than that of the REP-PCR (RP 6.20, MI 4.48). The REP-PCR and ERIC-PCR markers have been found to be useful for discrimination of B. thuringiensis isolates and reference strains. ERIC-PCR was the more informative of the two techniques. This study showed that the B. thuringiensis isolates collected from diverse habitats in India had a high degree of genetic diversity.     

Molecular characterization of Antarctic actinobacteria and screening for antimicrobial metabolite production

The present study aimed to isolate actinobacteria from soil samples and characterized them using molecular tools and screened their secondary metabolites for antimicrobial activities. Thirty-nine strains from four different location of Barrientos Island, Antarctica using 12 types of isolation media was isolated. The isolates were preceded to screening of secondary metabolites for antimicrobial and antifungal activities. Using high-throughput screening methods, 38% (15/39) of isolates produced bioactive metabolites. Approximately 18% (7/39), 18% (7/39), 10% (4/39) and 2.5% (1/39) of isolates inhibited growth of Candida albicans ATCC 10231^T, Staphylococcus aurues ATCC 51650^T, methicillin-resistant Staphylococcus aurues (MRSA) ATCC BAA-44^T and Pseudomonas aeruginosa ATCC 10145^T, respectively. Molecular characterization techniques like 16S rRNA analysis, Enterobacterial repetitive intergenic consensus—polymerase chain reaction (ERIC-PCR), Random amplified polymorphic DNA (RAPD) and composite analyses were used to characterize the actinobacteria strains. Analysis of 16S rRNA sequences is still one of the most powerful methods to determine higher taxonomic relationships of Actinobacteria. Both RAPD and ERIC-PCR fingerprinting have shown good discriminatory capability but RAPD proved to be better in discriminatory power than ERIC-PCR. Our results demonstrated that composite analysis of both fingerprinting generally increased the discrimination ability and generated best clustering for actinobacteria strains in this study.