Ishii Rep Pcr 2009

Environmental Microbiology (2009) 11(4), 733–740doi:10.1111/j.1462-2920.2008.01856.x Minireview Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity, ecology and evolution Satoshi Ishii1 and Michael J. Sadowsky2,3,4* 1 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan. 2 Department of Soil, Water, and Climate, 3 BioTechnology Institute and 4Microbial and Plant Genomics Institute, University of Minnesota, St. Paul, MN, USA. Summary A large number of repetitive DNA sequences are found in multiple sites in the genomes of numerous bacteria, archaea and eukarya. While the functions of many of these repetitive sequence elements are unknown, they have proven to be useful as the basis of several powerful tools for use in molecular diagnostics, medical microbiology, epidemiological analyses and environmental microbiology. The repetitive sequence-based PCR or rep-PCR DNA fingerprint technique uses primers targeting several of these repetitive elements and PCR to generate unique DNA profiles or ‘fingerprints’ of individual microbial strains. Although this technique has been extensively used to examine diversity among variety of prokaryotic microorganisms, rep-PCR DNA fingerprinting can also be applied to microbial ecology and microbial evolution studies since it has the power to distinguish microbes at the strain or isolate level. Recent advancement in rep-PCR methodology has resulted in increased accuracy, reproducibility and throughput. In this minireview, we summarize recent improvements in rep-PCR DNA fingerprinting methodology, and discuss its applications to address fundamentally important questions in microbial ecology and evolution. Introduction Microbial genomes contain a variety of repetitive DNA sequences, accounting for up to 5% of the genome (Ussery et al., 2004). Many of these repetitive DNA elements are of unknown function and have been localized to both intergenic and extragenic regions of the microbial genome. Recent data suggest that some of these repetitive elements may be involved in the synthesis or catabolism of RNA and DNA (Tobes and Ramos, 2005), or to be mediators of genome evolution (Schmidt and Anderson, 2006). These repetitive elements are typically comprised of duplicated genes, such as those encoding rRNA and tRNA, insertion sequence elements, transposons, short polynucleotide (3–7 mers) repeats, mosaic repetitive elements and other repetitive or palindromic sequences (Table 1) (Sadowsky and Hur, 1998; Versalovic and Lupski, 1998). The sizes of these interspersed repetitive elements vary from 15 bp (e.g. five-time repeats of trinucleotides) to hundreds of base pairs in length (e.g. RLEP element) (Versalovic and Lupski, 1998). Analysis of the whole genome sequences of many microbial species has revelled the presence of numerous repetitive sequences (Ussery et al., 2004; Tobes and Ramos, 2005). While some repetitive elements (e.g. HRS1 and RLEP) are specifically present in only a limited group of bacteria, others, such as the repetitive extragenic palindromic (REP) DNA sequences, have been found to be broadly distributed among many members of phylogenetically diverse bacteria (Sadowsky and Hur, 1998; Versalovic and Lupski, 1998; Tobes and Ramos, 2005). The use of repetitive elements for the analysis of bacterial genomes has proven to be a powerful tool for studies of microbial ecology, environmental microbiology, molecular diagnostics, medical microbiology and epidemiological analyses. Since each microbial strain or isolate has repetitive sequences located in distinct regions of their genome, the PCR technique, performed using sequences of these repetitive elements as primers and total genomic DNA as template, generates multiple amplicons that differ in size in direct proportion to the genomic Received 14 October, 2008; accepted 27 November, 2008. *For correspondence. E-mail [email protected]; Tel. (+1) 612 624 2706; Fax (+1) 612 625 2208. © 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd . time and cost. 2008).. 2008). but with better resolution. (1992) Hulton et al.. the improved quality of Taq DNA polymerase. MPTR. PFGE). 1994. the former technique can be performed with standard equipment and with less labour. 11. bacterial interspersed mosaic element. Frye and Healy. Rademaker et al. J. Given the usefulness of rep-PCR technique to genotypically characterize and track a large number of agriculturally. Other recent reviews concerning the rep-PCR technique are also available elsewhere (Sadowsky and Hur. 2008). pulsed-field gel electrophoresis. which can generate larger sized DNA fragments (~10 kb) than previous formulations. 2008). 1999).. ERIC. Hahm and colleagues (2003) compared rep-PCR. While several comparative analyses have shown that rep-PCR and PFGE have nearly equal discriminating power. The rep-PCR DNA fingerprinting technique has proven to be a valuable tool to identify. RSRjb STRR Repetitive sequence type Mosaic repetitive element Mosaic repetitive element Large repetitive element Insertion sequence Polynucleotide repeat Large repetitive element Insertion sequence Polynucleotide repeat Palindromic sequence Large repetitive element Insertion sequences Polynucleotide repeat Reference Gilson et al. STRR. but other closely worded synonyms can also be frequently found in the literature. (1990) Hermans et al. 1998). PFGE. Although other genomic DNA fingerprinting methods are also useful to group and identify microbes (e. (1993) Sharples and Lloyd (1990) Thierry et al. Rademaker et al. medically and environmentally important microorganisms.. enteric repetitive intergenic consensus. PFGE and multilocus sequence typing (MLST) had better discriminant power than did plasmid profiling and antimicrobial susceptibility testing when subtyping Salmonella enterica serovar Typhimurium strains obtained from various animals. (1991) Martin et al. intergenic repeat unit. Methodological considerations Several modifications of the standard rep-PCR technique have been investigated to increase accuracy. HRS1. 124–127 bp enterobacterial repetitive intergenic consensus (ERIC) sequences or the 154 bp BOX element as priming sites for PCR (Table 1) (Versalovic et al. Ishii and M. was shown by Chokesajjawatee and colleagues (2008) to produce repPCR DNA fingerprints that clustered Vibrio strains similar to what was found by using DNA:DNA hybridizations. Similarly. track and examine diversity among medically and environmentally important microorganisms (Sadowsky and Hur.. Following electrophoresis. multiplex-PCR. Versalovic et al. 1991. and more throughput than PFGE (Olive and Bean. Rademaker et al. (1984). Sadowsky Table 1. and (ii) describe recent applications of this tool that address fundamentally important questions in microbial ecology and microbial evolution. Environmental Microbiology. Versalovic et al. and concluded that rep-PCR and PFGE were the most discriminative DNA fingerprinting methods available. BIME. In contrast. and at the same time decrease time. the distribution of the resolved amplicons generates a genomic DNA fingerprint pattern unique for each bacterial strain or isolate (Rademaker et al. (1985) Mazel et al. distance between the binding sites of adjacent repetitive elements. The quality and ultimate usefulness of the DNA fingerprints generated by using rep-PCR are influenced by both the PCR reaction itself and the method(s) used to resolve the generated amplicons into discrete bands. Most rep-PCR-based DNA fingerprinting studies have used short polytrinucleotides.g. Tobes and Ramos (2005) Woods and Cole (1990) Kaluza et al. IRU. (1992) Stern et al. 1998. This technique has been referred to © 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.. hyper-reiterated sequences. short tandemly repeated repetitive sequences.. 35–40 bp REP sequences.. This technique is most often referred to as repetitive sequence-based PCR (Versalovic et al. 1998. 1998. On the level of the PCR reaction itself. reproducibility and throughput. DNA:DNA hybridizations. (1991) Judd and Sadowsky (1993) Doll et al. (1990) a.734 S. Commonly used for the rep-PCR DNA fingerprinting. 733–740 . ribotyping and AFLP to characterize Escherichia coli strains. 16S rRNA gene sequencing or ribotyping are more useful than rep-PCR in distinguishing bacteria at the generic or species level. 1991) or repetitive extragenic palindromic PCR (rep-PCR). the rep-PCR DNA fingerprinting technique is a simple and rapid method that has the necessary resolving power for microbial identification at subspecies or strain level (Versalovic et al. labour and cost associated with this technology. 1998. the objectives of this minireview are to: (i) summarize recent advances in rep-PCR DNA fingerprinting methodology. Selected endogenous repetitive sequences found in microbial genomes Sequence name BIME BOXa ERICa HRS1 (GTG)5a IRU IS6110 MPTR REPa RLEP RSRja. major polymorphic tandem repeat. 2006. Foley and colleagues (2006) reported that rep-PCR. such as (GTG)5. . Ishii and K. and the subsequent statistical analysis of the data. 1998).. 2008).. 2004). This reduced error due to inherent problems associated with gel-based electrophoresis.. and was capable of distinguishing among Mycobacterium tuberculosis and Mycobacterium avium complex strains with a resolution equivalent to IS6110-RFLP analysis (Cangelosi et al. 2008).. While several studies have used rep-PCR DNA fingerprinting to establish associations between particular microbial strains or pathovars with specific human. and genetically identical strains were found to have DNA fingerprints that were > 92–100% similar. 2007. While it is possible to analyse rep-PCR DNA fingerprinting data by eye when a sample set is relatively small and banding patterns are relatively simple. Rademaker et al. 2004. Recently. 2007).org. 2008). This technology has been successfully used to characterize and determine the potential sources of many thousands E. 2005). Johnson and colleagues (2004) reported the development of a horizontal. Most studies have used the GelCompar and BioNumerics (Applied Math. 1998. a free resource at http://www. 2005). Kortrijk. Since the internal size standard was applied in every lane of the gel. unpubl. 2007). Harrington et al. 11. this analysis is still subjective and labour-intensive (Rademaker et al. the technique has also proven useful to determine sources of microorganism and their transmission in disease outbreaks. 733–740 . This problem is amplified when large data sets are analysed and DNA fingerprints contain many closely spaced bands. and allow the user to determine correlations between banding patterns. 1998. This semi-automated rep-PCR system was reported to have discriminant power equal to PFGE when typing Streptococcus pneumoniae. The R program. Freeman et al. Frye and Healy. coli is currently used as an indicator of faecal contamination. 2001.. While the original FERP method was relatively expensive and required an automated system for fragment separation and detection.. which can be subsequently analysed using web-based DiversiLab software and libraries of fingerprints from several bacterial (Cangelosi et al. analysis of DNA fingerprint patterns. 1995. Vogel et al.. such as a capillary gel sequencing unit (Rademaker et al.. 2008). 2005. Freeman et al. GA) mycobacterial primer kit (Freeman et al. data). 2007). Applications of rep-PCR DNA fingerprinting for environmental microbiology The rep-PCR technique has been extensively used over the last decade for molecular epidemiological applications and to classify human and plant pathogens.. the potential source(s) of this bacterium in the environment is usually not known (Ishii and Sadowsky. 2008). Belgium) software packages which facilitate fingerprint acquisition and normalization.. Sources of non-disease-causing microorganisms have also been investigated by using rep-PCR DNA fingerprinting. a semi-automated rep-PCR DNA fingerprinting system has become commercially available. was identified by using rep-PCR DNA fingerprinting system and the Bacterial Barcodes (Athens. bias-free. animal or plant diseases (Versalovic et al. For more comprehensive analyses. fluorophoreenhanced rep-PCR (HFERP) technique that used conventional horizontal agarose gel electrophoresis coupled to the use of fluorescently labelled amplicons and fluorescently labelled internal size standards.. archaeal (Cleland et al. For example. each band in each DNA fingerprint pattern could be normalized to the internal standard.Rep-PCR DNA fingerprinting 735 as long-range rep-PCR (Brumlik et al. coli strains (Johnson et al. 2008). 2005... and clustering algorisms have been shown to increase the accuracy and reproducibility of analyses of multiple rep-PCR DNA fingerprinting data sets (S. the source of microbes responsible for a large tuberculosis outbreak among homeless persons in Seattle. The DiversiLab system (bioMérieux. (ii) fingerprint normalization and (iii) statistical analysis of fingerprint data. France) uses a microfluidics device to separate rep-PCR amplicon rapidly and reliably (Healy et al. clustering and discriminant analyses. including correspondence. Deficiencies in the resolution of gel-based electrophoresis systems have also been shown to limit the ability of rep-PCR DNA fingerprinting to differentiate among closely related or genetically near-identical strains (Johnson et al. For example..r-project. 2008). The fluorophore-enhanced repPCR (FERP) technique allows precise size calling of fluorescently labelled DNA fingerprint bands relative to fluorescently labelled internal DNA size markers applied in the same lane as the samples to be analysed (Versalovic et al.. Versalovic et al. while E.. Rademaker et al. draw dendrograms and perform a variety of statistical analyses. plant symbionts. Kadota. Environmental Microbiology. Chokesajjawatee et al. Computer-assisted DNA fingerprint acquisition and data analysis has also increased the accuracy of the rep-PCR DNA fingerprinting technique. 2004... Rep-PCR and HFERP DNA fingerprinting have both been used in an attempt to differ- © 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd. Ishii et al... and a large number of other environmentally and medically relevant microorganisms (see reviews by Sadowsky and Hur. Marcy e’Etoile. 2005). 2006. Computer-assisted rep-PCR DNA fingerprinting data analysis is described in more detail elsewhere (Rademaker and de Bruijn. Washington.. 2004. soil bacteria. Several free and commercially available computer programs have proven especially useful in the rapid. Ross et al. commercially available software is frequently used. Computer-assisted DNA fingerprint analysis involves three distinct steps: (i) fingerprint acquisition.. 2008) and eukaryote species (Wise et al. 736 S. Ishii and colleagues (2007) successfully applied HFERP DNA fingerprinting to determine the potential sources of E. and to ‘type’ Xanthomonas sp. After specificity testing. D and E) (Goullet and Picard. Ishii and co-workers also noted that there was not a direct correlation between HFERP DNA fingerprint patterns and phylogenetic groups among a larger set of E. 1 (S. Johnson. 733–740 . they are susceptible to changes over time caused by polymorphisms. coli clones in the environment (Byappanahalli et al. While initial studies performed using multilocus enzyme electrophoresis (MLEE) classified the ECOR strains into six phylogenetic groups (A.K. strains to the subspecies. coli clones can be found to overwinter in soils form northern Minnesota over multiple seasons. such as those in the ECOR collection (Ochman and Selander. Sadowsky... Ferguson and M. rep-PCR DNA fingerprinting appears to be a useful tool to analyse faecal contamination in environments containing a limited number of potential input sources (Ishii and Sadowsky. Similarly. DNA bands specific to species or strains of interests were isolated directly from gels (Giesendorf et al. Ohnishi et al. are often used for studies of microbial evolution and phylogeny. The HFERP DNA fingerprinting method has also been used to examine the presence. Helicobacter (Kwon et al. The overall basis for this approach is that the rep-PCR DNA fingerprint patterns of environmental E. Since all of these factors alter the genome structure of microorganisms. 1998). serotyping is based on variation in somatic (O). Sadowsky entiate among genetically near-identical. For example. and introgression of genomic DNA via horizontal gene transfer (Sadowsky and Hur. 2001. Microbial genome evolution While rep-PCR DNA fingerprint patterns are thought to be stable over many generations of microbial growth (Kang and Dunne. including disease-causing and -suppressive Streptomyces strains (Sadowsky et al. 2003). the PFGE (Ohnishi et al. recombination between repeated sequences. Sadowsky. 2008). 1997). the clonality of Bacteroides in faecal samples from humans who received a 7-day antibiotic treatment. prophage insertions. coli in knownsource libraries. while MLEE detects polymorphic variation in enzymes revealed following electrophoresis.. unpubl. genotypic distribution. but ecotypically distinct E.. Meyer and M. 1998). including species members within the genera Rhizobium and Bradyrhizobium (Sadowsky and Hur. The technique was found to be useful in differentiating E... 2006). This disparity extends beyond phylogenetic group classification. Ishii. For example. in spring samples. and the shift in population structure of acetic acid bacteria during wine fermentation have also been examined by using rep-PCR DNA fingerprinting (González et al. J. in part. J. 11. C.. 2005. effects of prophages (Metzgar et al. 2007). 2002) and HFERP (L.. Similarly. coli originating from treated wastewater.. Ishii and M.. coli populations obtained from multiple host animals. Ishii and colleagues (2006) reported that ‘indigenous’ soilborne E. 2004). it also has other uses. 1998) and Burkholderia (Matheson et al.. survival and persistence of specific E.A. 2000). coli samples should be similar to the fingerprints of E. or by cloning of rep-PCR products (Matheson et al. Similar trends were also reported for analyses performed using microsatellites (short-motif repeats) to characterize ECOR strains (Metzgar et al. the technique was also found to be useful in distinguishing among closely related plant pathogens. and the plasticity of microbial genomes. Moreover. these fragments can be useful as hybridization probes for detection of specific bacterial species or strains in environmental samples. and has proven to be useful to examine the phylogeny of Nitrobacter strains isolated from geographically and ecophysiologically different origins (Vanparys et al. The rep-PCR DNA fingerprinting technique has been used to examine genetic diversity among nitrogen-fixing bacteria. 1984). 1997). unpublished). coli O157:H7 serotype strains.J... 1989).. loss or rearrangement of indigenous plasmids. B2. In addition. 1996).. 2007). 1993.. K. 2000. Environmental Microbiology.P. which were present on the beach in the summer to fall months. 2004. 2002) or lack of sensitivity. 1998). 2001). 2006. coli contaminating a beach near Duluth. rep-PCR has been used to generate speciesand strain-specific DNA probes by targeting rep-PCR amplicons in Campylobacter (Giesendorf et al. coli originating from various animal host sources (Dombek et al. rep-PCR fingerprinting can be used to study microbial genome evolution and molecular phylogeny. coli isolates (n = 1531) obtained from humans and 12 animal sources. 2007). B1. Although the rep-PCR DNA fingerprinting technique has mostly been used as genotyping and strain-tracking tools... Ishii et al. an example of which is shown in Fig. In this case. Jernberg et al. coli in river water and sediment samples in south-central Nebraska.. 1993). Ishii et al. Escherichia coli reference strains. Kwon et al.. While large databases are necessary to adequately represent genotypic diversity in E. to the hypermutable microsatellites. This lack of correlation may be due.. strain or pathovar level (Rademaker et al. Johnson et al. Vogel and colleagues (2007) used automated rep-PCR (the DiversiLab system) and reported that cattle and wildlife were the main sources of E.J. due to its high resolving power. thus allowing identification of unknown isolates (Johnson et al. Minnesota. strains within a given phylogenetic group could not be clustered based on rep-PCR DNA fingerprinting patterns (Johnson and O’Bryan. 2005). from those coming from waterfowl. data) techniques have also revealed great genetic diversity among DNA fingerprinting of E. flagella (H) and capsular (K) antigens as © 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd. rapid and direct method to identify possible rearrangements. potentially due to prophage-mediated genome rearrangements. or compositional changes in microbial genome structure. These authors also observed the appearance of new rep-PCR DNA fingerprint patterns that were different from those of the initial 48 strains. coli populations by mixing 48 different strains with autoclaved manure slurry and used rep-PCR DNA fingerprinting to examine clonality of populations. 733–740 . Duriez and colleagues (2008) reconstituted E. They reported that virulence genes were lost from specific clonal populations during manure storage (within 3 weeks).. This is chiefly due to the fact that the rep-PCR technique has the sensitivity to accurately genotype microbes at the strain or isolate level. As such. directly detect changes in nucleotide base composition. such as rep-PCR and PFGE. Thus. Environmental Microbiology. detected by several immunological-based assays. Ten strains each were randomly chosen from a collection (n = 1531) of E. coli strains isolated from humans. Concluding remarks Although the majority of the rep-PCR DNA fingerprinting studies have been performed for epidemiological and strain tracking purposes. In contrast. 11. Phylogenetic groups were determined by using multiplex PCR as previously described (Clermont et al. 1. coli in the environment. indicating the emergence of new genotypes during manure storage. Relationships between DNA fingerprints were determined by using Pearson’s curve-based correlation coefficient and the dendrogram was constructed by using the UPGMA clustering method. they provide a simple.Rep-PCR DNA fingerprinting 737 Fig. since genome fingerprinting methods. coli strains obtained from humans and 12 animal sources. these methods use indirect gross changes in enzyme mobility or serological reaction to categorize strains. Dendrogram showing the relatedness of E. cows and sheep as determined by HFERP DNA fingerprint analysis performed using the BOXA1R primer. 2000). Rep-PCR DNA fingerprinting has been recently used to examine the loss of virulence genes from E. this technique also has great versatility and can be used to study microbial ecology and microbial evolution. suggesting that gene content within the populations were changing in response to the survival and/or adaptation to this new environment. it has © 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd. and Goldberg... (2003) Subtyping of foodborne and environmental isolates of Escherichia coli by multiplex-PCR. R. Zo. Lising. Hahm. Á.. Frye. supported by the NOAA Office of Sea Grant. D. Fedorka-Cray. ISME J 2: 233–241.J.L.G.... Ishii and M. and Colwell. Koeken. S. M. J Gen Microbiol 135: 135–143. J Clin Microbiol 31: 1541– 1546.. If the resulting fingerprints are reproducible. A. E.. (2008) Determination of clonality and relatedness of Vibrio cholerae isolates by genomic fingerprinting. 444–471.A.. B.. van Belkum. 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