Evaluation of a fluorescent amplified fragment length polymorphism (FAFLP) methodology for the genotypic discrimination of Aeromonas taxa

FEMS Microbiology Letters 177 (1999) 83^92

Evaluation of a £uorescent ampli¢ed fragment length polymorphism (FAFLP) methodology for the genotypic discrimination of Aeromonas taxa Geert Huys *, Jean Swings Laboratorium voor Microbiologie, Universiteit Gent, K.L. Ledeganckstr. 35, B-9000 Ghent, Belgium Received 17 April 1999; accepted 31 May 1999

Abstract A fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting assay is evaluated for its ability to differentiate DNA hybridization groups in the genus Aeromonas. After empirical determination of optimal assay conditions using a limited set of strains, 98 well-characterized type and reference strains encompassing all known Aeromonas taxa were subjected to FAFLP fingerprinting using the standardized protocol. The present study clearly indicates that the use of fluorescent dye-labeled primers does not significantly affect the high capacity of this technique to differentiate among genotypically closely related Aeromonas taxa. Compared to the original AFLP protocol involving the application of radioisotopes, the new FAFLP technology offers a better performance when considering speed of analysis and user safety. On the other hand, FAFLP fingerprints exhibited a significant reduction in the relative number of bands compared to the corresponding autoradiographic patterns. In our hands, the omission of the preselective amplification step and the use of a size standard mix enhanced the cost effectiveness and the reproducibility of the technique. Cluster analysis of FAFLP band patterns generated from Aeromonas type and reference strains demonstrated once more the high correlation of AFLPgenerated data with DNA-DNA homology data. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Aeromonas; Fluorescent ampli¢ed fragment length polymorphism ; Bacterial taxonomy; DNA ¢ngerprinting

1. Introduction Members of the genus Aeromonas are Gram-negative rod-shaped organisms that typically occur in various freshwater environments worldwide [1]. In addition, aeromonads have been frequently isolated

* Corresponding author. Tel.: +32 (9) 2645249; Fax: +32 (9) 2645092; E-mail: [email protected]

from various foods and clinical specimens [2,3]. Over the years, well-documented studies have clearly demonstrated that these bacteria can produce a wide variety of virulence factors possibly playing an important role in the generation of extra-intestinal and gastro-intestinal infections [3,4]. Because of their relevance as primary animal pathogens and opportunistic human pathogens, the correct and reliable identi¢cation of clinical and environmental Aeromonas isolates is becoming increasingly important. As a

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 9 5 - 5

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result of the marked disagreement between phenotypic and genetic data, the present Aeromonas taxonomy allows identi¢cation of unknown isolates at two levels, i.e. the phenospecies level (de¢ned on the basis of phenotypic characteristics) and the genomospecies or DNA hybridization group (HG) level (de¢ned on the basis of DNA homology studies). Currently, at least 14 phenospecies and 15 HGs have been described in Aeromonas (Table 1). The fact that some of these phenospecies comprise several HGs (e.g. HGs 4, 5A, and 5B in Aeromonas caviae) whereas other HGs are dispersed among di¡erent phenospecies (e.g. HG3 encompasses Aeromonas salmonicida and Aeromonas hydrophila-like strains) clearly illustrates the complex nature of Aeromonas systematics and stresses the need for highly discriminatory identi¢cation methods. At present, DNA ¢ngerprinting techniques are considered to be highly valuable tools for the highresolution di¡erentiation of Aeromonas isolates. Methods like randomly ampli¢ed polymorphic DNA (RAPD) analysis [5,6] and pulsed-¢eld gel electrophoresis (PFGE) [7,8] are very useful for individual typing of Aeromonas isolates but are either too sensitive to be used in genomic species discrimination or have not yet been tested throughout the entire genus. Analysis of sequence polymorphisms in ribosomal DNA (ribotyping) has been successfully used for both epidemiological and taxonomic investigations in Aeromonas [9,10]. Recently, Borrell and coworkers [11] reported that restriction fragment analysis of PCR-ampli¢ed 16S rRNA genes is also a promising tool for the identi¢cation of Aeromonas species. However, the rather limited number of bands per pattern may restrain the potential of the latter two methods to discriminate among certain Aeromonas sp. pairs. In addition, digital capture and computerized analysis of the obtained data has not yet been evaluated for both techniques. At this moment, ampli¢ed fragment length polymorphism (AFLP) analysis can be considered one of the most discriminating genomic methods to distinguish among Aeromonas HGs. Originally developed for plant breeding purposes, this technique essentially interweaves the speci¢city of whole-genome restriction fragment analysis and the selectivity of high-stringency PCR ampli¢cation without prior knowledge of primer target sequences [12]. The

Table 1 Aeromonas strains included in this study Phenospecies

HGa Strain no.b

A. hydrophila

1

A. bestiarum

2

A. hydrophila

3

A. salmonicida subsp. salmonicida subsp. achromogenes subsp. masoucida subsp. smithia A. caviae

3 3 3 3 4

A. caviae

5A

A. caviae

5B

A. media

5B

A. eucrenophila

6

A. sobria A. veronii biovar sobria

7 8

A. jandaei

9

A. veronii biovar veronii 10 A. encheleia

11

A. schubertii

12

A. trota

13

A. allosaccharophila

NDc

A. enteropelogenes A. ichthiosmia A. popo¤i

ND ND ND

LMG 2844T , 13656, LMG LMG 13444, 13447, LMG LMG 13449, 13453, LMG

LMG 13439, 13658, LMG LMG 13446, 13448, LMG LMG 13451, 13674, LMG

LMG 13660 LMG 13662 LMG 13675

LMG 3780T LMG 14900T LMG 3782T LMG 16264T LMG 3775T , LMG 13454, LMG 13456, LMG 13457, LMG 13676, LMG 13678, LMG 13679, LMG 13680 LMG 13460, LMG 13461, LMG 13681, LMG 13683, LMG 13684 LMG 13465, LMG 13467, LMG 13468 LMG 9073T , LMG 14687, LMG 14688, LMG 14689 LMG 3774T , LMG 13057, LMG 13058, LMG 13060, LMG 13687 LMG 3783T , LMG 13469 LMG 13067, LMG 13068, LMG 13070, LMG 13071, LMG 13072, LMG 13073, LMG 13074, LMG 13693, LMG 13694,LMG 13695, LMG 13700 LMG 12221T , LMG 13064, LMG 13065, LMG 13066, LMG 13077, LMG 13079 LMG 9075T , LMG 16183, LMG 16332, LMG 16333, LMG 16334 LMG 13075, LMG 13076, LMG 16328, LMG 16329, LMG 16330T , LMG 16331, LMG 13061, LMG 13062, LMG 13691 LMG 9074T , LMG 12655, LMG 12668, LMG 13473 LMG 12223T , LMG 13080, LMG 13081, LMG 13082, LMG 13083 LMG 14021, LMG 14058, LMG 14059T LMG 12646T LMG 12645T LMG 17541T , LMG 17542, LMG 17543, LMG 17544, LMG 17545, LMG 17546, LMG 17547

T, type strain. a HG, hybridization group b LMG, Culture Collection of the Laboratorium voor Microbiologie Gent, Universiteit Gent, Belgium. c ND, not determined.

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AFLP method is currently used in various ¢elds of microbiology including taxonomy [13,14], epidemiology [15,16], population ecology [17,18], and molecular evolution [19]. In the genus Aeromonas, it has been demonstrated that AFLP clustering and HG delineation are usually highly concordant and that AFLP data can be used for the construction of a reliable taxonomic framework in nomenclatural rearrangements [20] and new species descriptions [21]. As a result, AFLP analysis has been successfully used for the classi¢cation and identi¢cation of aquatic and clinical Aeromonas isolates [22,23] as well as for clonal analysis of Aeromonas populations [24]. From a pure technical point of view, however, it is clear that the original AFLP technique is not very well suited for rapid identi¢cation or routine diagnostics due to the use of radioactively labeled PCR primers and the time-consuming preparation of PCR submixes. In order to reduce its complexity and to increase the speed of analysis, the AFLP protocol was recently modi¢ed by the introduction of £uorescent dye-labeled primers and through the application of an automated DNA sequencer for data capture. So far, this adapted non-radioactive method has been successfully evaluated for the identi¢cation and/or typing of Acinetobacter [25], Streptococcus [26], Campylobacter [27], and Cryptosporidium [28] strains. In this study, we evaluated the usefulness of the £uorescent AFLP (FAFLP) protocol implemented on the ABI Prism1 377 DNA Sequencer (PE Applied Biosystems) for the di¡erentiation of DNA hybridization groups in Aeromonas. Following optimalization of speci¢c technical parameters, an extended collection of Aeromonas type and reference strains representing all known taxa in this genus was subjected to AFLP ¢ngerprinting using a standardized protocol. Results obtained by the numerical analysis of digitized £uorescent ¢ngerprints were compared with the existing classi¢cation based on autoradiographically generated AFLP patterns.

2. Materials and methods 2.1. Bacterial strains and culture conditions For this study, a collection of 98 genotypically

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well-characterized type and reference strains representing all known Aeromonas taxa was included (Table 1). All strains were previously subjected to AFLP ¢ngerprinting analysis using the radioactive protocol [29]. Aeromonas strains were grown on trypticase soy agar (TSA) medium containing 30.0 g l31 trypticase soy broth (BBL, Cockeysville, MD, USA) and 15.0 g l31 Bacteriological Agar No. 1 (Oxoid, Basingstoke, Hampshire, UK) for 24 h at 28³C except for A. salmonicida strains which were cultured at 22³C. The Plesiomonas strain was grown at 37³C. 2.2. DNA isolation and AFLP template preparation The extraction and puri¢cation of total genomic DNA was based on the rapid guanidinium thiocyanate extraction method of Pitcher and co-workers [30]. The electrophoretic quality check on agarose and the spectrophotometric determination of the DNA concentration were performed as described previously [29]. AFLP templates were prepared from approx. 1 Wg high-molecular-mass genomic DNA through double enzymatic digestion using the endonucleases ApaI and TaqI subsequently followed by restriction halfsite-speci¢c ligation of doublestranded oligonucleotide adapters and selective precipitation according to Janssen et al. [12]. The adapters were prepared by mixing equimolar amounts of the partially complementary oligonucleotides 5P-TCGTAGACTGCGTACAGGCC-3P and 5P-TGTACGCAGTCTAC-3P (for ApaI) and 5P-GACGATGAGTCCTGAC-3P and 5P-CGGTCAGGACTCAT-3P (for TaqI). 2.3. PCR reactions In order to determine optimal PCR conditions, it was tested whether the addition of a preselective ampli¢cation step might contribute to the quality and the reproducibility of the FAFLP band patterns as suggested in the protocol of the AFLP1 Microbial Fingerprinting method (PE Applied Biosystems). In this step, restriction fragments are ampli¢ed using unlabeled primer sequences designed on the basis of the adapter and the remaining restriction site sequences. Consequently, a low-level selection is obtained where all fragments comprising modi¢cations at both ends will be ampli¢ed exponentially as op-

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posed to the one-end-modi¢ed fragments that will only amplify linearly. All PCR primers used in this study were purchased from Amersham Pharmacia Biotech; their designations were given according to Janssen et al. [12]. PCR reactions were routinely performed in sterile 500-Wl Eppendorf tubes using the Genius thermocycler (Techne, Cambridge, UK). For one preselective ampli¢cation reaction, 4.0 Wl precipitated template solution (containing approx. 40 ng DNA) was added to a mix of 0.5 Wl primer A00 (1 WM; 5P-GACTGCGTACAGGCCC-3P), 0.5 Wl primer T00 (5 WM; 5P-CGATGAGTCCTGACCGA3P), and 15 Wl Ampli¢cation Core Mix comprising PCR bu¡er, nucleotides, and AmpliTaq0 DNA polymerase (PE Applied Biosystems). Primers A00 and T00 contained no additional nucleotides as selective bases at their 3P ends. The following temperature program was used for preselective ampli¢cation: (i) initial extension at 72³C for 2 min and (ii) 20 cycles of denaturation at 94³C for 20 s, annealing at 56³C for 30 s, and extension at 72³C for 2 min. In between subsequent experiments, PCR products were stored at 4³C. The selective ampli¢cation step was performed in two ways, i.e. either using the preselective ampli¢cation product (see Section 2.2) or the original AFLP templates as PCR template. In this way, it was possible to compare FAFLP band patterns generated with one-step PCR (selective) and two-step PCR (preselective-selective) procedures. Before entering the selective ampli¢cation stage, preselective PCR product solutions were diluted 20 times in T0.1E bu¡er (10 mM Tris-HCl, 0.1 mM EDTA; pH 8.0) whereas the original AFLP templates were used undiluted. Next, 1.5 Wl DNA template was added to a mix containing 0.5 Wl primer A01-6FAM (1 WM; 5P6FAM-GACTGCGTACAGGCCCA-3P), 0.5 Wl primer T01 (5 WM; 5P-CGATGAGTCCTGACCGAA3P), and 7.5 Wl Ampli¢cation Core Mix. Primer A016FAM contains the blue £uorescent ABI Prism dye 6-FAM (PE Applied Biosystems). Both primers used in selective ampli¢cation contained an adenosine (underlined) at their 3P ends as selective base. The following thermal cycler parameters were programmed: (i) initial denaturation at 94³C for 2 min, (ii) 10 cycles of denaturation at 94³C for 20 s, annealing using a decreasing stringency rate at [663(n31)] ³C for 30 s with n = the cycle number,

and extension at 72³C for 2 min, (iii) 20 cycles of denaturation at 94³C for 20 s, annealing at 56³C for 30 s, and extension at 72³C for 2 min, and (iv) ¢nal extension at 60³C for 30 min. 2.4. Sample preparation Following selective ampli¢cation, PCR products were diluted three times with TE bu¡er (10 mM Tris-HCl, 1 mM EDTA; pH 7.6). According to the AFLP1 Microbial Fingerprinting method implemented on the ABI Prism 377 DNA sequencer (PE Applied Biosystems), optimal electrophoretic conditions are obtained when using a speci¢c loading bu¡er (LB) mix. This LB mix contains a commercially available size standard which serves as an internal reference necessary for the proper alignment of patterns in the GelCompar normalization program. Samples are prepared by mixing 1 Wl of PCR product dilution with 2 Wl of LB mix containing 1.25 Wl deionized formamide, 0.25 Wl blue dextran/50 mM EDTA loading solution (supplied with the size standard), and 0.5 Wl GeneScan(GS)-500 [ROX] size standard (PE Applied Biosystems). In this study, however, the yellow £uorescent ABI Prism dye TAMRA was used instead of the recommended red £uorescent ROX dye because the former label generated higher signal intensities during preliminary trials. In order to obtain the optimal conditions for the alignment of sample peaks during the GelCompar normalization step (see below), we compared the use of a mix of two size standards against the use of only one (i.e. GS-500) size standard. The suggested size standard mix comprised 0.5 Wl GS-500 [TAMRA], 0.5 Wl GS-2500 [TAMRA], 0.25 Wl loading solution, and 0.75 Wl formamide. Samples were stored at 4³C and loaded within 2 h after preparation. 2.5. Polyacrylamide gel electrophoresis Selective ampli¢cation products were separated on a 4.25% denaturing polyacrylamide gel on an ABI Prism 377 automated DNA sequencer (PE Applied Biosystems). The gel was prepared by mixing 5.3 ml of 40% acrylamide-bisacrylamide stock solution (19:1) (Bio-Rad, Hercules, CA, USA), 18 g urea (¢nal concentration 6 M) (Bio-Rad), and 5.0 ml

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10UTBE bu¡er (1 M Tris-HCl, 1 M boric acid, 20 mM EDTA; pH 8.3) and ¢nal adjustment to 50 ml with HPLC-grade water (Merck). Prior to adding the TBE bu¡er, the mixture was deionized by adding 0.5 g AG0 501-X8 Resin (Bio-Rad) and stirring for 10 min. Finally, the deionized mixture was degassed for 10 min by vacuum ¢ltration. To 50 ml of gel solution, 250 Wl of 10% ammonium persulfate (BioRad) and 25 Wl of N,N,NP,NP-tetramethyl-ethylenediamine (Bio-Rad) were added. Gels were poured using an ABI 377 gel cassette with 0.2-mm spacers and a square-tooth comb, and were allowed to polymerize for 2^4 h at room temperature. After mounting the gel cassette on the ABI Prism 377 automated sequencer, the transparency of the laser detection route was inspected by the Plate Check command and the gel matrix was prerun to 51³C. Prior to loading volumes of 2.2 Wl, the samples were heated at 95³C for 3 min and then quickly cooled on ice. Electrophoresis was done in 1UTBE bu¡er for 2.5 h at 51³C. 2.6. Data processing and numerical analysis After tracking and extraction using the GeneScan analysis software (PE Applied Biosystems), FAFLP lanes were saved as individual sample ¢les. For each extracted sample lane, fragment sizing was performed by generating a sizing curve based on the prede¢ned electrophoretic fragment distribution of the internal size standard (i.e. GS-500 or a mix of GS-500/GS-2500). For numerical analysis, data intervals were delineated between the 50- and 400-bp bands of the internal size standard. Sized electropherograms were imported into the GelCompar1 software (Applied Maths BVBA, Kortrijk, Belgium) using the ABICON program (Applied Maths BVBA) with resolution for conversion set at 1000. The resulting FAFLP sample lanes were corrected for differences in electrophoretic mobilities by GelCompar1 Doublegel Normalization against the comigrated size standard tracks. The following userde¢ned settings were used: resolution 500 points, smoothing factor 3 points, background subtraction using the rolling disk principle, and with automatic rescaling. Comparative analysis of normalized sample tracks was performed by GelCompar1 Analysis software based on the Pearson product-moment cor-

87

relation coe¤cient and UPGMA clustering algorithms.

3. Results and discussion 3.1. Optimalization of the standard protocol In this study, we evaluated the usefulness and the taxonomic resolution of the FAFLP ¢ngerprinting methodology for the classi¢cation of all Aeromonas taxa described so far. Clearly, the signi¢cant reduction in hands-on time is the main advantage of the £uorescent AFLP1 Microbial Fingerprinting method (PE Applied Biosystems) when compared to the original radioactive protocol. More speci¢cally, AFLP analysis of bacterial genomes can now be performed much faster due to the omission of radioisotope labeling, autoradiography, and radioactive waste disposal, the shorter gel polymerization time, the use of a commercially available PCR premix, and the implementation of denaturing polyacrylamide electrophoresis and data capture on the ABI Prism 377 DNA sequencer. Using the optimalized FAFLP protocol (see below), a complete experiment from the DNA template level to numerical data analysis can essentially be carried out in 1.5 days whereas a similar set up according to the radioactive method would take at least 3 days depending on the time needed for autoradiographic exposure. The e¡ect of omitting the preselective ampli¢cation in the protocol of the AFLP1 Microbial Fingerprinting method was investigated with the purpose of shortening the time needed to complete the PCR step and to save a signi¢cant amount of consumables without reducing the discriminatory power and reproducibility of the technique. For this evaluation, DNA templates of six Aeromonas strains (i.e. LMG 13662, LMG 13061, LMG 14687, LMG 13679, LMG 14059, and LMG 13073) were used in two separate PCR assays, one performing both preselective and selective ampli¢cation and one including only selective ampli¢cation. In order to allow proportionate data comparison, all resulting amplicons were included in one single electrophoresis run. Numerical analysis of the resulting FAFLP band patterns clearly showed that ¢ngerprints generated without preselective ampli¢cation were of equal in-

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Fig. 1. Electropherogram of the GeneScan-500/2500 size standard mix run under denaturing conditions as described in this study. From left to right, the 24 reference peaks correspond to DNA fragments with the following size (in bp): 35, 50, 75, 94, 100, 109, 116, 139, 150, 160, 172, 186, 200, 222, 233, 238, 250, 269, 286, 300, 340, 350, 361, and 400. Underlined fragment sizes originate from the GeneScan-500 size standard.

tensity and sometimes displayed more bands compared to the patterns produced with preselective ampli¢cation (data not shown). Although we could not ¢nd a reasonable explanation for this di¡erence, it was decided to leave out the preselective ampli¢cation step in further AFLP studies because FAFLP patterns generated without preselective ampli¢cation generally exhibited good reproducibility (see below). The main advantage of this adaptation in the protocol was that PCR ampli¢cation and electrophoresis could now be performed in one day. Secondly, we also investigated the bene¢cial e¡ect of using a mix of two size standards (i.e. GS-500 and GS-2500) instead of only the GS-500 size standard in obtaining the most accurate normalization possible. Although the GelCompar normalization concept is a very powerful tool for alignment of sample patterns, it is obvious that much of its success strongly depends both on the choice of the standard reference and on the critical eye of the user. Providing that bands are evenly distributed along the length of the gel track, a higher number of bands in a reference track should theoretically result in a more accurate normalization result. Preliminary experiments showed that the majority of the FAFLP amplicons were between 35 bp and 400 bp in size. In this range, the GS-500 size standard displays 13 fragments that can be de¢ned as reference positions whereas an equimolar mixture of GS-500 and GS-2500 exhibits a total of 24 suitable reference peaks (Fig. 1). For tracks derived from a single gel, UPGMA clustering analysis using the Pearson product-moment correlation coe¤cient demonstrated that the minimum cor-

Fig. 2. Simpli¢ed dendrogram showing cluster analysis of FAFLP band patterns generated from 98 Aeromonas type and reference strains (Table 1). The dendrogram was constructed with the UPGMA clustering method using the Pearson productmoment correlation coe¤cient.

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relation levels of the GS-500 reference tracks were up to 4% lower compared to the corresponding values for the mixed GS-500/2500 reference tracks. This can be explained by the fact that the relatively large size intervals of 50 bp in the GS-500 chromatogram are ¢lled up in the GS-500/2500 mixed pattern with extra reference peaks originating from the GS-2500 chromatogram (Fig. 1). Especially for typing purposes in clinical and ecological studies, the use of the GS-500/ 2500 instead of GS-500 is de¢nitely recommended to allow optimal correction for size-independent electrophoretic mobility shifts. 3.2. Reproducibility Using the radioactive AFLP methodology for genotypic analysis of aeromonads, the level of reproducibility was determined by including the FAFLP pattern of a well-chosen Aeromonas strain as a reference track after every ¢fth sample lane during each electrophoresis run [29]. Following normalization and background subtraction, it was found that the intragel-speci¢c correlation levels for the reference samples were between 95.0% and 98.0% as generated by UPGMA clustering. In the FAFLP protocol, on the other hand, each sample lane includes a mix of two molecular size standards serving as an internal reference. When considering GS-500/2500 reference tracks, we found that minimum intragel-speci¢c correlation levels were generally in the range of 92.0^ 95.0% when using the UPGMA clustering method. Because no apparent deviations in band position alignment could be observed visually, this relatively lower correlation may in part be caused by the lower number of bands in the £uorescent standard reference compared to the autoradiographic reference and because of relative sensitivity of the Pearson correlation coe¤cient to non-linear intensity variations in individual GS-500/2500 reference tracks. In some cases, the problem of variable peak height can be avoided by choosing band-based similarity coef¢cients that solely rely on the presence or absence of a prede¢ned band. From our experience, however, it appeared that autosearch band assignment algorithms frequently failed to discriminate between sample bands and background signals making this type of coe¤cient even less suitable than the Pearson index. Interestingly, also Kokotovic and On [27] re-

89

cently found that peak heights among their FAFLP pro¢les were variable upon repeat. According to the authors, however, this variability did not a¡ect the conclusions relating to the genotypic relationships of the strains under study. Other microbial studies that also used the FAFLP technology did not report on pattern reproducibility in terms of similarity values [25] or were restricted to comparison of GeneScan software-derived electropherograms [26]. Compared to the radioactive AFLP methodology, the use of £uorescent size standards as internal references has the main advantage that the reference pattern is not subjected to variations in PCR conditions. In this way, band alignment and gel normalization are thus only in£uenced by electrophoretic parameters. In turn, the user can no longer check the stability and the reproducibility of the PCR process among subsequent FAFLP experiments which is still very relevant in relation to reliable database management. Therefore, we recommend including the AFLP template of a group-speci¢c strain as a so-called PCR reference on every gel within a given study. Using template from A. veronii strain LMG 9075T as a PCR reference, we found that its FAFLP pattern systematically displayed a constant band distribution indicating that PCR conditions were kept stable during this entire study. 3.3. Classi¢cation of Aeromonas taxa A comparison between Aeromonas ¢ngerprints generated by the radioactive and the FAFLP protocols clearly demonstrated a signi¢cant di¡erence in the number of bands per track (data not shown). AFLP patterns derived from autoradiograms usually displayed 40^50 bands in the size range 80^550 bp whereas the corresponding ¢ngerprints extracted from £uorescent electropherograms contained between 25 and 35 fragments in the size range 50^400 bp. However, the majority of the Aeromonas strains included in this study displayed a unique FAFLP band pattern which still indicates the highly discriminating capacity of this ¢ngerprinting method. From the FAFLP patterns shown in the study of Koeleman and co-workers on genomic species of Acinetobacter [25], no apparent di¡erence in band number between corresponding ¢ngerprints generated by radioactive and £uorescent AFLP technologies could

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be observed. Because the majority of the templates used in this study were generated during the course of previous studies [29], we investigated whether the observed decrease in relative band numbers might be caused by the partial degradation of these DNA templates as a result of repeated thawing and freezing. In this regard, it was found that FAFLP patterns obtained from freshly prepared templates of duplicate cultures of several strains from Table 1 were identical to the corresponding ¢ngerprints generated from old templates. Consequently, the possibility that the reduction in relative band number was due to DNA template degradation was excluded. Clearly, further experience with the FAFLP technology is needed to verify whether a decrease in the number of bands may a¡ect the discriminatory power of this technique in microbial typing studies. At a delineation level of 35% Pearson product-moment similarity, a total of 14 AFLP clusters could be de¢ned when using the UPGMA clustering method. The simpli¢ed dendrogram depicted in Fig. 2 clearly shows that there is a good overall correlation between AFLP clusters and Aeromonas HGs delineated by DNA-DNA hybridizations. Moreover, a comparison with the previously published Aeromonas dendrogram obtained from autoradiographic AFLP patterns [29] did not reveal major di¡erences in cluster delineation, and this despite the slightly lower linkage levels of the reference tracks and the decrease in band variability observed with the FAFLP methodology. The type strain of the species Plesiomonas shigelloides, an organism that has historically always been closely associated with aeromonads, clearly falls out of the major Aeromonas cluster delineated at a correlation level of 10%. As expected, the taxonomic problems reported in previous AFLP studies on Aeromonas [29] were again highlighted in Fig. 2, e.g. the tight grouping of the A. enteropelogenes and A. ichthiosmia type strains in AFLP clusters of A. trota and A. veronii, respectively, and the inability to genotypically di¡erentiate the three known A. allosaccharophila reference strains from the species A. veronii. However, the majority of the AFLP clusters solely comprised representatives of one single Aeromonas HG and could therefore be de¢ned as reference entries in the generation of an identi¢cation database (AEROLIB) using the library creation options o¡ered by GelCompar1 Analysis. In this way,

the AEROLIB database can be used for the identi¢cation of unknown Aeromonas isolates that could not be classi¢ed with faster but less discriminatory phenotypic methods. 3.4. Conclusions In the present study, it was demonstrated that the implementation of the AFLP methodology on an automated DNA sequencer using £uorescent dye-labeled primers did not in£uence the high discriminative power of this DNA ¢ngerprinting method for the classi¢cation of genotypically closely related Aeromonas taxa. Compared to the original AFLP protocol involving the application of radio-isotopes, the new FAFLP technology has many advantages in terms of analysis speed and user safety. On the other hand, FAFLP ¢ngerprints displayed a signi¢cant decrease in relative band number in comparison with the corresponding autoradiographic patterns, thus stressing the need for further evaluation of the described FAFLP methodology towards individual strain characterization. In addition, the current investigation pointed out that the elimination of the preselective ampli¢cation step and the application of a size standard mix were bene¢cial towards the cost e¡ectiveness and the reproducibility of the described method. Cluster analysis of FAFLP band patterns obtained from a collection of well-characterized Aeromonas type and reference strains again expressed the high correlation between AFLP groups and DNA hybridization groups.

Acknowledgements This research was carried out in the framework of FAIR-CT96-1703 of the European Commission.

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