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.     

An evaluation of ERIC PCR and AP PCR fingerprinting for discriminating Salmonella serotypes

PCR fingerprints of 89 Salmonella isolates belonging to 22 serotypes were obtained using ERIC PCR (enterobacterial repetitive intergenic consensus PCR) and AP PCR (arbitrarily primed PCR) to evaluate the ability of different fingerprinting methods to differentiate or identify serotypes and subtypes. Fingerprints were scored and comparisons were made using a computer program. ERIC PCR produced a unique, complex fingerprint for almost every isolate, but these fingerprints did not identify serotypes. One AP PCR primer also produced complex fingerprints that discriminated among isolates, but again did not identify serotypes. A second AP PCR primer produced simple patterns, including one pattern shared by 35 isolates from 12 different serotypes. In general, the three sets of PCR fingerprints distinguished isolates, but were not correlated with serotypes. Matching fingerprints from different gels by computer was difficult, since similarities were based on both intense and faint bands. In addition, this study suggests that dendrograms created from PCR fingerprints should be viewed with caution.     

Evaluation of genetic diversity among Pseudomonas citronellolis strains isolated from oily sludge-contaminated sites

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.     

Genetic relationships among strains of Xanthomonas fragariae based on random amplified polymorphic DNA PCR, repetitive extragenic palindromic PCR, and enterobacterial repetitive intergenic consensus PCR data and generation of multiplexed PCR primers useful for the identification of this phytopathogen.

Genetic relationships among 25 isolates of Xanthomonas fragariae from diverse geographic regions were determined by three PCR methods that rely on different amplification priming strategies: random amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) PCR, and enterobacterial repetitive intergenic consensus (ERIC) PCR. The results of these assays are mutually consistent and indicate that pathogenic strains are very closely related to each other. RAPD, ERIC, and REP PCR assays identified nine, four, and two genotypes, respectively, within X. fragariae isolates. A single nonpathogenic isolate of X. fragariae was not distinguishable by these methods. The results of the PCR assays were also fully confirmed by physiological tests. There was no correlation between DNA amplification product patterns and geographic sites of isolation, suggesting that this bacterium has spread largely through exchange of infected plant germ plasm. Sequences identified through the RAPD assays were used to develop three primer pairs for standard PCR assays to identify X. fragariae. In addition, we developed a stringent multiplexed PCR assay to identify X. fragariae by simultaneously using the three independently derived sets of primers specific for pathogenic strains of the bacteria.     

Assessment of genetic diversity of Iranian Ascochyta rabiei isolates using rep‐PCR markers

Genetic diversity and population structure among 29 isolates of Ascochyta rabiei (AR) obtained from diseased chickpea plants in six different geographical origins in Iran was characterized by MAT and rep‐PCR (BOX/ERIC/REP) markers. Both mating types were found in all six populations, and the frequencies of mating types were variable between populations. The majority of the isolates belonged to Mat1‐1 (58.12%) with the remainder (41.88%) being Mat1‐2. A dendrogram was calculated with Jaccard's similarity coefficients with unweighted pair group method clustering (UPGMA) for the combination of rep‐PCR results, AR strains were differentiated into four clusters (A–D) at 60% similarity level. ERIC, REP and BOX showed a total of 19, 37 and 24 alleles per locus, respectively. Gene diversity (He) and Shannon's information index (I) were the highest in the REP (He = 0.82; I = 2.11), while the lowest values were estimated for the ERIC (He = 0.42; I = 1.3). Our result showed that among the three techniques studied, REP‐PCR produced the most complex amplified banding patterns, which reflected a high degree of diversity among the Iranian AR strains. ERIC‐PCR was the least discriminating method, and BOX‐PCR was intermediate. To the best our knowledge, this is first study of assessment of genetic diversity of AR isolates by rep‐PCR markers.     

Characterization of serogroup A Neisseria meningitidis strains by rRNA gene restriction patterns and PCR: Correlation with the results of serotyping, subtyping and multilocus enzyme electrophoresis

Abstract We studied 35 strains of Neisseria meningitidis serogroup A from different locations (France, Central African Republic, Sudan and Burkina Faso) using both ribotyping and a polymerase chain reaction (PCR). A non-radioactive probe label was used for ribotyping; detection consisted of an immunoenzymatic procedure using a bispecific antibody. The PCR was designed to amplify the 16S–23S rDNA internal transcribed spacer. These techniques were compared with other markers. The strains were identified as belonging to three clones (I, III-1, IV) by multilocus enzyme electrophoresis (MEE) and to three subtypes by serological methods. Ribotyping identified five groups and PCR identified four groups. Ribotyping gave more diversity between strains than either MEE or sero/subtyping, but confirmed the epidemiological data provided by the combination of these two techniques. The PCR provided a simple and convenient one-step procedure for the differentiation of strains of serogroup A.     

Evaluation of Genetic Diversity among Pseudomonas citronellolis Strains Isolated from Oily Sludge-Contaminated Sites

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.     

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.     

用rep—PCR技术研究中国花生根瘤菌的多样性

采用细菌基因组重复序列ＰＣＲ技术（简称ｒｅｐＰＣＲ）中常用的ＲＥＰＰＣＲ和ＥＲＩＣＰＣＲ,对从中国１１个省、市的２３个点、２４个花生品种采集的根瘤中分离的５９株花生根瘤菌Ｂｒａｄｙｒｈｉｚｏｂｉｕｍｓｐ．（Ａｒａｃｈｉｓ）进行多样性研究,同时对来自国外的６株花生根瘤菌及１４株参比慢生根瘤菌也进行了比较。得到的低相似性结果表明中国花生根瘤菌基因组存在显著的多样性。ＲＥＰＰＣＲ揭示,在相似性５０％上分为１１个群,而ＥＲＩＣＰＣＲ却得到２４个分群。这两种结果对菌株的分群有差异,暗示这两种短重复序列在慢生根瘤菌基因组中的分布的不同。没有发现菌株间基因组的多样性分布与花生品种、地理来源之间的必然联系。将两者电泳图谱结合并分析,得到介于上述两者间的结果。此结果进一步反映了菌株基因组间存在的多样性。同时还表明ｒｅｐＰＣＲ不仅是研究生物多样性的快速简便方法,还可应用于菌株的鉴别和生态学研究。     

Intraspecies discrimination of Lactobacillus paraplantarum by PCR

Lactobacillus paraplantarum is a species phenotypically close to Lactobacillus plantarum. Several PCR methods were evaluated to discriminate L. paraplantarum strains and among them, a PCR using an enterobacterial repetitive intergenic consensus (ERIC) sequence differentiated L. paraplantarum from other Lactobacillus species. In addition, a combination of ERIC and random amplified polymorphic DNA (RAPD) analysis distinguished among seven strains of L. paraplantarum tested. ERIC-PCR profiles showed several strain-specific DNA fragments in L. paraplantarum, among them, a 2.2-kb ERIC marker, termed LpF1, found to be specific to strain FBA1, which improved the skin integrity in an animal model. The LpF1 encodes three proteins similar to Lactobacillus fermentum AroA, TyrA, and AroK, which are involved in the shikimate pathway. A primer pair specific to FBA1 based on the internal sequence of LpF1 amplified a 950-bp FBA1-specific fragment LpF2. Southern blot analysis of Dra I-digested genomic DNA of L. paraplantarum strains using LpF2 as a probe showed that LpF2 is distinctive of strain FBA1 among 16 L. paraplantarum strains. Because both ERIC- and RAPD-PCR are fast and technically simple methods, they are useful for the rapid discrimination of L. paraplantarum strains and for the development of new strain-specific DNA markers for identifying industrially important strains.     

Use of Rep‐PCR for Genetic Diversity Analyses in Fusarium culmorum

Fusarium culmorum is a pathogen of economically important grain crops. In this work, Rep‐PCR was used to identify genetic diversity in F. culmorum isolates which have been collected from wheat fields in Turkey. Reproducible genomic fingerprints were amplified in each strain by PCRs of prokaryotic repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC) and BOX sequences. Totally 104 molecular markers were evaluated and similarity comparisons were shown as a dendrogram. The average genetic diversity was 52.3% ranging from 15.8% to 88.7% according to the Rep‐PCR data. Cluster analysis showed agreement with the distance of sampling locations. The highest genetic similarity (84.2%) was determined between two F. culmorum isolates (F1 and F2) originated from the same agro‐ecological region. Our results showed that Rep‐PCR is convenient and rapid for genetic diversity analyses and strain differentiation in F. culmorum.     

Application of PCR ribotyping and tDNA-PCR for Klebsiella pneumoniae identification

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90.6% of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.     

新疆土著大豆根瘤菌种群遗传结构的初步分析

应用重复序列REP（repetitive extragenic palindromic,重复基因外回）和ERIC（ente-robaterial repetitive intergenic consensus,肠细菌重复基因间共有序列）结合聚合酶链式反应（ERP－PCR和ERIC－PCR）对从新疆有集的27株土大豆根瘤菌染色体进行指纹分析,发现在相似水平0.5时可基本分为两大聚类群,一个类群主要包括慢生型根瘤菌,另一类群为快生型根瘤菌,来自同一地区的根瘤菌具有较高的遗传相似性,以上结果表明该技术中对大豆根瘤菌进行种群结构和遗传多样性分析的有效手段。     

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.     

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.     

Rapid identification of pseudomonas avellanae field isolates,causing hazelnut decline in central italy,by repetitive PCR genomic fingerprinting

Pseudomonas avellanae is the main cause of hazelnut (Corylus avellana L.) decline, the so called ‘moria’, in central Italy where it has already killed more than 30 000 trees. Its current identification is very long requiring biochemical, physiological and nutritional tests as well as pathogenicity tests and takes not less than 6 months for its completion. In the present study the reliability of the repetitive polymerase chain reaction (rep‐PCR) technique for a rapid and accurate identification of such a pathogen was compared with the traditional identification method. In order to assess the variability of the pathogen, REP, BOX and ERIC primer sets were used in preliminary work to generate genomic fingerprints of 60 P. avellanae reference strains previously isolated from different areas of hazelnut cultivation. ERIC primers yielded the most discriminative clustering of strains that were grouped according to their geographic origin. Sixty field isolates collected from hazelnut orchards of central Italy, planted with different cultivars, during the years 1996–98 were submitted to either the traditional identification methods or to rep‐PCR by using ERIC primers. The latter technique accurately identified all the isolates that were also identified by the traditional methods. Whole‐cell protein analysis by means of sodium dodecyl sulphate‐polyacrylamide gel electrophoresis confirmed this achievement. Rep‐PCR can be successfully adopted for the rapid and accurate identification of P. avellanae in central Italy and it constitutes a very useful tool for the sanitation of the area.     

细菌基因组重复序列PCR技术及其应用

细菌中散在分布的DNA重复序列近年来不断被报道,基因外重复回文序列和肠细菌基因间共有重复序列是两个典型的原核细胞基因组散在重复序列。重复序列在染色体上的分布和拷贝数具种间特异性,用它们的互补序列作为引物,以细菌基因组DNA为模板进行PCR扩增反应,反应产物的琼脂糖电泳可以提供非常清晰的DNA指纹图谱,使用此图谱既可对各种微生物进行快速分型及鉴定,又可对它们进行DNA水平上的遗传多样性分析。细菌基因组重复序列PCR技术具有简捷、快速、结果稳定等特点,可对细菌进行分子标记,用于菌株分型、分类鉴定和亲缘关系等方面的研究。     

Identification of Saccharomyces sensu stricto and Torulaspora yeasts by PCR ribotyping

18S rDNA + ITS1 and 25S rDNA PCR products covering more than 95% of the nuclear ribosomal DNA repeat unit of 28 Saccharomyces sensu stricto and Torulaspora yeasts and their anamorph forms were digested with Hae III, Msp I, Hinf I and Cfo I. Using combinations of two restriction enzymes, specific ribotyping patterns of six species were found. PCR ribotyping offers a convenient tool for quick identification of yeast isolates, but specificity of ribotyping patterns should be checked with a larger number of strains to avoid misidentification because of lack of variation within different taxa or because of strain-specific ribotyping patterns of species type strains.     

Genetic Relatedness among Environmental, Clinical, and Diseased-Eel Vibrio vulnificus Isolates from Different Geographic Regions by Ribotyping and Randomly Amplified Polymorphic DNA PCR

Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificus isolates.     

Genetic basis of enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprint pattern in Sinorhizobium meliloti and identification of S. meliloti employing PCR primers derived from an ERIC-PCR fragment

The enterobacterial repetitive intergenic consensus (ERIC)-PCR method was employed to generate genomic amplification products of Sinorhizobium meliloti strain 2011. Eleven distinctive PCR fragments obtained in PCR reactions by using the ERIC2 primer were cloned and their partial or complete nucleotide sequences established. DNA sequences that extended past the ERIC2 primer region were not conserved among the 11 PCR fragments and showed no sequence similarity to the enterobacterial ERIC consensus sequence. Thus, repetitive ERIC or ERIC-like sequences seem not to be an integral part of the S. meliloti genome. An amplification product of S. meliloti 2011 was identified which was present in S. meliloti strains but absent in other rhizobial species. Based on the nucleotide sequence information, a pair of PCR primers was designed and used for PCR amplification of sequences of S. meliloti laboratory strains 2011, L5–30, AK631 and 102F34. Nucleotide sequence analysis of the amplification products revealed a 100% DNA sequence conservation. Database searches showed that the DNA fragment putatively encodes the C-terminal part of a protein displaying similarity to 2-hydroxyacid dehydrogenases of various organisms. The newly designed PCR primers should be useful for the rapid identification of S. meliloti isolates. Received: 17 February 1999 / Accepted: 9 April 1999