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.     

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.     

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.     

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.     

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.     

Molecular typing of colonizing Streptococcus agalactiae strains by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) in a Chennai based hospital

Streptococcus agalactiae is reported to be an asymptomatic vaginal colonizer in Indian women, although it is considered one of the major causes of neonatal infections in many European countries. DNA based molecular typing methods are more reliable than the conventional serotyping method for identification and typing of this pathogen. In the present study, we have evaluated genetic diversity among colonizing S. agalactiae strains (n=86) by using a PCR-based genotyping method i.e. Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR). With ERIC-PCR fingerprinting at 60% similarity level in a dendrogram generated by UPGMA cluster analysis, 10 different ERIC groups were identified, which were subdivided into 62 distinct genotypes at ≥ 95% similarity level. Based on these findings, we demonstrate that ERIC-PCR is a simple, rapid, and inexpensive tool with sufficient discriminatory power and is applicable for characterization and genotyping of a large number of clinical isolates of S. agalactiae at molecular level.     

Assessment of the genetic diversity of Xylella fastidiosa isolated from citrus in Brazil by PCR-RFLP of the 16S rDNA and 16S-23S intergenic spacer and rep-PCR fingerprinting

The genetic diversity among twenty three strains of Xylella fastidiosa, isolated from sweet orange citrus, was assessed by RFLP analysis of the 16S rDNA and 16S-23S intergenic spacer and by rep-PCR fingerprinting together with strains isolated from coffee, grapevine, plum and pear. The PCR products obtained by amplification of the 16S rDNA and 16S-23S spacer region were digested with restriction enzymes and a low level of polymorphism was detected. In rep-PCR fingerprinting, a relationship between the strains and their hosts was observed by using the BOX, ERIC and REP primers. Two major groups were obtained within the citrus cluster and relationships to the geographic origin of the strains revealed. Citrus strains isolated from the States of São Paulo and Sergipe formed one group and strains from the Southern States formed another group. Distinct origins of X. fastidiosa in the Southern and Southeastern States is postulated. The pear isolate was distantly related to all of the other X. fastidiosa strains.     

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.     

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.     

Identification and inter-relationship analysis of Bradyrhizobium japonicum strains by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD)

Genomic DNA of 13 Bradyrhizobium japonicum strains was prepared and analysed by restriction fragment length polymorphism (RFLP) with nif and nod probes, and by random amplified polymorphic DNA (RAPD) with 11 primers of arbitrary nucleotide sequence. Polymorphism was observed in both analyses. The RFLP and RAPD banding patterns of different strains were used to calculate genetic divergence and to construct phylogenetic trees, allowing studies on the relationships between the strains. RFLP with nif and nod probes permitted the separation of the strains into two divergent groups, whereas RAPD separated them into four main groups. RAPD allowed closely related strains to be distinguished.     

Variation revealed by RAPD‐PCR profiles of microsymbiont Anabaena azollae strains from four N2 fixing Azolla cultures

Nitrogen fixing Anabaena azollae strains isolated from four different Azolla cultures were characterized based on their total protein profile and RAPD profile to study the existing variation among them. As expected, the isolates showed almost similar protein banding patterns, but exhibited differences in 40–70 KDa protein subunits. Polymerase chain reaction of the DNA of the isolates, using four different primers, amplified specific sequences of DNA and showed clear polymorphism among the isolates. The RAPD profile generated the fingerprinting pattern characteristic of each strain based on the sequence of the primers used. Common band sharing observed between the strains A. azollae‐RS‐KK‐SK‐AM and A. azollae‐RS‐KK‐SK‐RP probably represents maternal inheritance of DNA to the progeny. The polymorphic bands were generated specifically for the isolates A. azollae‐RS‐KK‐SK‐RP and A. azollae‐RS‐KK‐SK‐AM with primers numbered 2 and 4, respectively, which could be developed as possible markers for these isolates.     

Typing of clinical and environmental strains of Aeromonas spp. using two PCR based methods and whole cell protein analysis

Two PCR based typing methods i.e. random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR were evaluated for typing of 42 Aeromonas isolates from clinical and environmental sources and whole cell protein (WCP) profiles were analyzed. Both RAPD and ERIC-PCR showed a high level of genetic diversity. Numerical index of the discriminatory (D) values were 0.94 and 0.96 (>0.90) for RAPD and ERIC-PCR, respectively. No correlation in banding pattern and evidence of genetic similarity was found between Aeromonas isolates from environmental and clinical sources. Therefore these techniques are highly reproducible and sensitive methods for typing the Aeromonas isolate from different sources. WCP profile showed two major variable regions i.e. 20 kDa to 45 kDa region and 70 kDa to 85 kDa region. Though WCP profiling had less discriminatory power, use of this method in combination with other established typing methods such as RAPD and ERIC-PCR may be helpful for reliable typing of Aeromonas isolates or to identify new proteins with pathogenic potential.     

23株不同地理来源嗜酸硫杆菌的系统发育及其遗传差异

【目的】研究不同地理来源嗜酸硫杆菌的系统发育及其遗传差异,以及基因指纹图谱技术聚类与嗜酸硫杆菌地理来源的相关性。【方法】采用16S-23S r RNA间隔区(ITS)序列建立系统发育树,并结合ERIC和BOXAIR两种引物进行rep-PCR,以及rus基因扩增,对不同地理来源嗜酸硫杆菌进行分析。【结果】分离自不同样点的23株嗜酸硫杆菌遗传差异显著,依据ITS序列系统发育树被划分为5个大类群,与rep-PCR指纹图谱的分类结果较为接近,其中Acidithiobacillus ferrooxidans在ITS系统发育和BOXAIR-PCR指纹聚类分析中被划分为2个类群,但在ERIC-PCR中归为1个类群,rus基因分组中,在系统发育和聚类分析中处于同一类群的菌株拥有不同类型的rus基因,说明嗜酸硫杆菌的亚铁氧化途径与系统发育类群无明显相关性;ITS基因拥有区分近缘种或亚种的能力,且BOXAIR-PCR的分辨能力较强,非常适于嗜酸硫杆菌的遗传差异分析。     

Genotyping by RAPD-PCR analyses of Malassezia furfur strains from pityriasis versicolor and seborrhoeic dermatitis patients

Malassezia furfur is lypophilic yeast commonly associate with dermatological disorders. In the present work, we described the isolation of 47 M. furfur strains from three groups of patients: pityriasis versicolor (21 isolates), seborrhoeic dermatitis (15 isolates) and seborrhoeic dermatitis of the HIV positive patients (11 isolates). To investigate the identity of the strains at molecular level, DNA genomic of M. furfur strains were prepared and used to RAPD-PCR analyses. RAPD assay were carried out using two decamer primers and bands pattern generated were analyzed by an Unweighted Pair-Group Method (UPGMA). Dendrogram established a distinct differentiation between M. furfur isolates from pityriasis versicolor and seborrhoeic dermatitis patients with or without AIDS. We concluded that RAPD typing presented a high discriminatory power between strains studied in this work and can be applied in epidemiological investigation of skin disease causing by M. furfur.     

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.     

Comparison of PCR fingerprinting techniques for the discrimination of <Emphasis Type="Italic">Salmonella enterica</Emphasis> subsp. <Emphasis Type="Italic">enterica</Emphasis> serovar Weltevreden isolated from indigenous vegetables in Malaysia

Salmonella enterica subsp. enterica (S.) serovar Weltevreden has emerged as a public health problem in many countries. Genomic DNA of S. Weltevreden from indigenous vegetables namely ‘selom’ (Oenanthe stolonifera), ‘pegaga’ (Centella asiatica), ‘kesum’ (Polygonum minus) and ‘kangkong’ (Ipomoea aquatica) were characterized by duplex-polymerase chain reaction (duplex-PCR), multiplex-polymerase chain reaction (multiplex-PCR), random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results demonstrated that a total of four clusters and three single isolates were generated from ERIC-PCR with primers ERIC-1 and ERIC-2 whereas RAPD with arbitrary primers OPAR2, OPAR17 and OPAR19 discriminated the S. Weltevreden into nine clusters and eight single isolates at a common 65% similarity level with discriminatory index (D) of 0.7443 and 0.9394 respectively. Composite analysis of banding profiles generated from RAPD-PCR and ERIC-PCR showed eight clusters and six single isolates at 65% similarity level with the highest D value that is 0.9508. On the other hand, PCR-RFLP and duplex PCR data exhibited a consistent profile for S. Weltevreden. Multiplex-PCR targeting three different antibiotic resistance genes and a common Salmonella specific gene segment produced two distinguishing profiles among the S. Weltevreden examined. These results demonstrated that the combined analysis of RAPD-PCR and ERIC-PCR is a better tool for characterizing S. Weltevreden than individual methods.     

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.     

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.     

Use of <Emphasis Type="Italic">rpoB</Emphasis> sequences and rep-PCR for phylogenetic study of <Emphasis Type="Italic">Anoxybacillus</Emphasis> species

This study was conducted to investigate the applicability of rpoB, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA gene sequence similarity analysis in the thermophilic genus Anoxybacillus. Partial rpoB sequences were generated for the 14 type strains of Anoxybacillus species and 6 other strains of four Anoxybacillus species. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The rpoB gene was found to provide a better resolution for Anoxybacillus species, with lower interspecies sequence similarities. The rpoB sequence similarity analysis permitted a more accurate discrimination of the species within the Anoxybacillus genus than the more commonly used 16S rRNA gene. Furthermore, rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP-, ERIC-, and BOX-PCR) were employed for the specimens of genus Anoxybacillus. Through comparison of the three methods, it was found that the BOX-PCR method generated more informative results than REP-PCR for the studied strains; BOX-PCR profiles were more distinct for the different strains, including a higher number of bands. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (rep-PCR) constitute a suitable molecular approach for the validation and maintenance of taxonomy within the Anoxybacillus genus. The results of this study show that rpoB and rep-PCR provide rapid and reliable methods for molecular typing of Anoxybacillus species.     

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.