Taxonomic Diversity of Pseudomonads Revealed by Computer-interpretation of Ribotyping Data

System. Appl. Microbiol. 19,541-555 (1996) © Gustav Fischer Verlag

Taxonomic Diversity of Pseudomonads Revealed by Computerinterpretation of Ribotyping Data ROLAND BROSCH, MARTINE LEFEVRE, FRANCINE GRIMONT, and PATRICK A. D. GRIMONT Unite des Enterobacteries, Unite INSERM 389, Institut Pasteur, 75724 Paris Cedex 15, France

Received July 22, 1996

Summary In the framework of a European project entitled "High Resolution Automated Microbial Identification", the ability of rRNA gene restriction pattern determination (ribotyping) to uncover the taxonomic diversity of a collection of bacteria was tested on 226 strains of Pseudomonas sensu lato. Improved ribotyping methodology included automated DNA extraction and purification, low voltage electrophoresis, vacuum transfer of restricted DNA fragments to nylon membranes, hybridization with a chemically labeled probe in a hybridization incubator, computer capture of restriction patterns with automatic migration measurements, fragment size interpolation, and comparison of patterns using programs of the Taxotron package (Institut Pasteur, Paris, France). Two endonucleases were used (Smal and HindI). The strains, belonging to more than 40 nomenspecies, displayed 169 unique rRNA gene restriction patterns with Smal, and 159 unique rRNA gene restriction patterns with HincH. A combined analysis of both restriction data yielded 79 ribogroups or isolated ribotypes. Most (92.7%) ribogroups were homogeneous with respects to nomenspecies. Some nomenspecies were split in several ribogroups (e. g. P. putida, P. (luorescens, P. marginalis, P. pseudoalcaligenes). Nomenspecies represented by single strains gave isolated ribotypes. Biovars of P. (luorescens and P. putida formed distinct ribogroups. However, P. marginalis pathovars were not separated by ribotyping. When the ribotyping data (ribogroups) were compared to published phenotypic data (phenons) obtained with Biotype-lOa strips (BioMerieux, La Balme-Ies-Grottes, France) and Biolog GN Microplates (Biolog, Hayward, CAl, an exclusive phenon-ribogroup correspondence was observed for 13 Biotype-lOa phenons/ribogroups and for 14 Biolog phenons/ribogroups. Other Biotype-lOa or Biolog phenons showed partial correspondence with ribogroups or were split into several ribogroups. Most unclustered strains in phenotypic analyses were also unclustered in the ribotyping study. DNA-DNA hybridization was used to verify some species delineation. Ribogroup R38 which contained most of P. (luorescens biovar I strains was found to constitute a DNA hybridization group distinct from P. (luorescens strains in other ribogroups or biovars. The type strain of P. (luorescens, which was unclustered by ribotyping, was found isolated by DNA relatedness. Ribogroup R1 which contained most of P. putida biovar A strains was found to constitute a DNA relatedness group distinct from biovar B strains. A total of 30 isolates from polluted environment were included in the study and 9 of them were identified to six known taxons. Thirteen other isolates constituted five ribogroups not represented by a reference strain and seven isolates gave isolated ribotypes. This work showed the identification potential of ribotyping and the need to extend the ribotype database. It is now clear that the ribotype carries taxonomic information in addition to typing information.

Key words: DNA - Pseudomonas - Ribotyping - Environment

Introduction Molecular methods are now essential for determining the precise taxonomic organization of the bacterial world. Comparison of ribosomal ribonucleic acid (rRNA) sequences, or of corresponding gene sequences, by hybrid-

ization (De Vos and De Ley, 1983; Palleroni, et al. 1973) or alignment of determined nucleotide sequences (Fox et al., 1980) allows phylogenetic reconstruction. The taxonomic information thus obtained is invaluable above spe-

542

R. Brosch, M. Lefevre, F. Grimont, and P. A. D. Grimont

cies level. For species delineation, quantitative DNA-DNA hybridization is presently the most straightforward method (Wayne et a., 1987). Identification consists in the assignment of an isolate to a described bacterial species. When no guess can be made with respects to the taxonomic position of an isolate, rRNA sequencing is the most straightforward method although the resolution of the method may not be sufficient to separate closely related species (Stackebrandt and Goebel, 1994). When phenotypic properties or rRNA sequence comparison point to a group of closely related species, DNA-DNA hybridization, nucleic acid probe hybridization, or amplification of a species-specific DNA target are usually definitive identification methods. However, these methods are tedious when the initial identification guess is too vague (e. g. "a pseudomonad") thus implying that too many probes, amplification primers, or reference DNA samples are likely to be tested. Ten years ago, rRNA gene restriction patterns were proposed as a taxonomic tool for specific and infraspecific characterization (Grimont and Grimont, 1986). Subsequently, the method, named ribotyping (Stull et aI., 1988) was mostly used for differentiation at infraspecific and infrasubspecific levels, i. e. for epidemiological purposes (Irino et aI., 1988; Koblavi et aI., 1990; Stull et aI., 1988). Only a few taxonomic applications (identification at species level) were published (Grimont et aI., 1989). In the course of a European project on High-Resolution Automated Microbial Identification (HRAMI), the taxonomically complex genus Pseudomonas sensu lato was chosen as material for the evaluation of various taxonomic methods. It was found essential that, among the methods to be evaluated, one might emerge as able to handle rather large numbers of strains and be faster than quantitative DNADNA hybridization. Since it seemed that both typing and taxonomic information might be found in rRNA gene restriction patterns (ribotypes), the aim of the present work was to find out whether ribotyping was able to uncover the taxonomic diversity of the genus Pseudomonas sensu lato. This genus has now been split into several genera and a concise overview of the present taxonomy of this group of bacteria is given by Kersters et al. (1996). In the course of the work, automatic DNA extraction and computer interpretation of restriction patterns were systematically used.

Materials and Methods

Bacterial strains The 226 strains studied are listed in Figure 1. A total of 196 strains belonging to 40 species were obtained from the Laboratorium voor Microbiologie Gent Culture Collection (LMG), Ghent, Belgium. The origin of most of these strains can be found in the LMG catalogue (1992) or on the Internet (http://www.belspo.be/bccrn/db/selbccm.htm). Strains were grown following the recommendations of the culture collection, and stored at -80°C in brain-heart infusion supplemented with 50% (vol./vol.) glycerol. Thirty isolates (LMG 13958 to 13961, LMG 13963 to 13983, LMG 13984 to 13988) originated from Gesellschaft fur

Biologische Forschung (Braunschweig, Germany). They were recovered from water and sediment samples of a highly polluted tributary of the river Elbe in Sachsen-Anhalt, Germany, and were isolated on media containing various xenobiotic compounds. These strains, referred to as Elbe-river isolates, were grown on Tryptocasein-soya agar (Diagnostics Pasteur, Marnes-IaCoquette, France) at 30°C and then stored at -80°C. The isolates were received as Pseudomonas sp.

Ribotyping 1. Bacterial DNA preparation Each strain was subcultured aerobically on a Tryptocaseinsoya agar plate overnight, checked for purity, harvested in 2 ml of lysis-buffer (Tris-HCI 0.05 M, EDTA 0.05 M, NaCi 0.1 M, pH 8), and lysed by adding sodium dodecylsulfate (SDS) and pronase (Calbiochem, La Jolla, CAl. The mixture was incubated 1-2 hat 60°C to allow cell lysis. DNA was extracted and purified using an Autogen 540 automated DNA-extraction system (AutoGen Instruments, Beverly, MA) using two phenol/chloroform purification steps, ethanol precipitation and suspension in TE buffer (Tris 0.01 MlEDTA 0.001 M). 2. Restriction analysis Purified bacterial DNA (3 to 5 !!g) were cleaved by restriction endonucleases SmaI and HindI following the supplier's instructions (Amersham International, Amersham, UK). Restriction fragments were separated by electrophoresis in 0.8% (wtlvol) horizontal agarose gel (Appligene, IlIkirch, France) in Tris-borate buffer (89 mM Tris, 89 mM boric acid, 2 mM disodium EDTA, pH 8.3) for 16 hat 1.5 V/cm. DNA of Citrobacter diversus 32, cleaved by MluI was included on each gel (4 lanes in a 20-lane gel) as molecular weight standard. The DNA fragments were transferred to a nylon membrane (Hybond-N, Amersham) using a VacuGene system (Pharmacia LKB Biotechnology, Uppsala, Sweden). Hybridization of DNA fragments on nylon membranes with an acetylaminofluorene (AAF)-Iabeled 16 + 23S rRNA probe (Eurogentec, Seraing, Belgium), reaction with mouse antiAAF monoclonal antibodies and anti-mouse IgG alkaline phosphatase-conjugated antibodies, and band visualization by 5bromo-4-chloro-3-indolyl phosphate (Sigma, St Louis, MO) were done as published (Grimont and Grimont, 1995).

Computer interpretation of ribotyping data Ribotyping banding patterns were scanned with One-Scanner (Apple Computers, Cupertino, CAl and then interpreted using various programs of the Taxotron package (Institut Pasteur, Paris, France) written for this project by one of us (PADG) to work on a color Macintosh computer (Apple). The package contained seven programs, four of which being relevant to the present study. These were RestrictoScan for lane and band detection and migration measurements, RestrictoTyper for interpolation of fragment sizes from migration values using the Schaffer and Sederoff (1981) algorithm, Adanson for clustering, and Dendrograf for drawing dendrograms. The following steps were taken to ensure the highest quality of the results: (1) interpolation of fragment size at lane position Lx was calculated with the experimental formula obtained for the nearest standard lane on the left (MI) and for the nearest standard lane on the right (Mr), both interpolated values (SI and Sr, respectively) being averaged using a weight related to lane proximity (weight of SI is the absolute difference between Lx and Mr, weight of Sr is the absolute difference between Lx and MI); (2) each standard lane fitting curve was closely examined to verify that all data points were on the curve; (3) for each scanned membrane, the schematic representation of bands drawn by Restric-

Ribotyping of Pseudomonas toTyper was visually compared to the actual patterns of the membrane to detect any missing or artifactuous band. Program RestrictoTyper compared pairs of patterns and calculated a distance coefficient which was the complement of the Dice index (Sneath and Sokal, 1973). First, a tolerance function (percent tolerated error) had to be set which could be a fixed value or a linear function of the fragment size. We chose to set a fixed value of 4%, indicating that two fragments were considered identical if their sizes did not differ by more than 4%. The distance coefficient was calculated as the number of nonmatching fragments divided by the total number of bands in both patterns. A distance matrix was then generated for patterns obtained after cleavage by each endonuclease (SmaI and HindI). These distance matrices were averaged using program Adanson and treated by the average linkage algorithm of Bartelemy and Guenoche (1988). This algorithm is a modification of the Unweighted Pair Group Method of Averages (Sneath and Sokal, 1973) making the clustering method insensitive to the order of strains in the distance matrix. The program generated a tree description file which was used by program Dendrograf to draw a dendrogram and produce a small order file. The order file was used by RestrictoTyper to reorder the fragment size files and produce a schematic graph showing the patterns in the same order as the strains in the dendrogram. The dendrogram and schematic graphs were saved as PICT files and assembled in a single picture by use of a commercial drawing program (Claris Works was used in this work). DNA-DNA hybridization

DNA relatedness among 65 selected Pseudomonas strains, including 35 strains received as P. fluorescens, and 14 strains received as P. putida was evaluated by the S1 nuclease method following a published procedure (Grimont et al., 1980). Pseudomonads being characterized by a high G+C content of their DNA (62-66 mol% according to Palleroni, 1984), hybridizations were carried out at the optimal reassociation temperature of 70°e. The temperature (Tm) at which 50% of the reassociated molecules are dissociated has been determined (Crosa et al., 1973). The difference (LlTm) between the Tm of the homoduplex and that of the heteroduplex is a measure of divergence (Brenner, 1978). In addition, 16 unidentified Elbe-river isolates with unique ribotypes were subjected to DNNDNA hybridization with labeled DNA from P. fluorescens and P. putida.

Results

Ribotyping Digestion of genomic DNA with Sma I and hybridization with 16S and 23S rRNA generated 169 unique ribotypes (Figure 1). With HindI, 159 unique ribotyping patterns were observed (Figure 1). The number of rRNA gene restriction fragments ranged from 4 to 13 for Sma I patterns and from 1 to 12 for HindI-patterns. Average linkage clustering of combined ribotype data yielded 79 ribogroups and isolated ribotypes designated Rl to R79 (Fig. 1). To fit other data (see discussion), ribogroup R53 was split in four groups with strains LMG 2839 and 6397 forming group R53a; strains LMG 11199 and 2332 forming group R53b; strains LMG 2243 and 5838 forming group R53c; and strains LMG 1228, 2334, and 6394 forming group R53d.

543

DNA relatedness DNA relatedness of 35 P. fluorescens strains to P. fluorescens strain LMG 1794 (type strain, biovar I), strain LMG 14569 (biovar I), and strain LMG 1244 (biovar III) are shown in Table 1. Thirty two strains representing five biovars and 15 ribogroups were 13 to 50% related to the type strain. Eleven strains of biovar I, ribogroup R38 were 63 to 82% related to strain LMG 14569 (biovar I, ribogroup R38) with ~ Tm values ranging from 0.0 to 5.0 dc. Two other biovar I strains displaying ribogroups R42 and R63 were 42 and 47% related to strain LMG 14569. Twenty-one strains of other biovars were 14 to 48% related to strain LMG 14569. Strain LMG 14576 (biovar 3, ribogroup R23 was 81 % related to strain LMG 1244 (biovar 3, ribogroup R23). Six other strains of biovar III and ribogroups R41, R44, and R64 were 46 to 51 % related to strain LMG 1244. Twenty-seven strains belonging to other biovars and ribogroups were 15 to 55% related to strain LMG 1244. DNA relatedness of 13 P. putida strains to P. putida LMG 2257 (type strain, biovar A) are shown in Table 2. Six strains of biovar A, ribogroup Rl were 79 to 95% related to strain LMG 2257. Seven strains of P. putida biovar B, representing ribogroups R7 and R35, were 18 to 29% related to strain LMG 2257. DNA relatedness of 16 strains of Pseudomonas sp. (Elbe-river isolates) to P. putida LMG 2257 and P. fluorescens LMG 14569 are shown in Table 3. The strains were only 15 to 44% related to P.fluorescens LMG 14569. Strain LMG 13977 (ribogroup Rl) was 69% related to P.putida LMG 2257 with a ~Tm value of 5.0 DC. Strains LMG 13964 and 13959 (ribogroups R2 and R4, respectively) were 57 to 61 % related to strain LMG 2257 with ~ Tm values of 6.0 and 7.0 DC, respectively. All other strains were 12 to 48% related to P. putida LMG 2257.

Discussion

Ribotyping Strains displaying similar SmaI patterns mostly also displayed closely related HincH rRNA gene restriction fragments. However, SmaI was more discriminative than HincH, as it generated more ribotypes than HincH. Gruner et al. (1993), reported that SmaI was not sufficiently discriminatory for ribotyping P. aeruginosa strains isolated from an intensive care unit. In our hands, SmaI discriminated Pseudomonas strains very well, since the seven strains of P. aeruginosa tested displayed five SmaI ribotypes and only two different HindI ribotypes. However, for some groups of strains (e. g. P. marginalis), HindI patterns were more heterogeneous than Sma I patterns. • Combined numerical study of ribotyping data. In the following ribogroups, the strains showed identical or almost identical patterns both with SmaI and HindI, R2 (Pseudomonas sp.), R5 (Pseudomonas sp.), R8 (Pseudomonas sp.), RIO (P. corrugata), R12 (P. syringae), R14 (P. caricapapayae), R15 (Burkholderia cepacia), R16 (P. meliae), R17 (P. ficuserectae), R18 (P. amygdali), R23 (P. fluorescens bv. III), R26 (P. tolaasii), R28 (Pseudomo-

R. Brosch, M. Lefevre, F. Grimont, and P. A. D. Grimont

544

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signation numbers of LMG strains in other culture collections are listed in the catalogue of the Belgian Coordinated Collections of Microorganisms - BCCM - or may be retrieved via the internet site http://www.belspo.be/bccmldb/selbccm.htm); P. = Pseudomonas; P. margo = Pseudomonas marginalis; pv = pathovar; R. = Ralstonia; T = type strain. " non-authentic strains, evidenced also by other methods (Grimont et aI., 1996; Vancanneyt et aI., 1996). 36

System. Appl. Microbiol. Vol. 19/4

R. Brosch, M. Lefevre, F. Grimont, and P. A. D. Grimont

546 1.0 i

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Fig. 1. Continued.

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Ribotyping of Pseudomonas 1.0

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548

R. Brosch, M. Lefevre, F. Grimont, and P. A. D. Grimont

Table 1. DNA relatedness among Pseudomonas {luorescens strains Present name

P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P.

{luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens {luorescens

Strain designation

LMG 14569 LMG 14564 LMG 5830 LMG 14562 LMG 14565 LMG 5825 LMG 14567 LMG 14563 LMG 14570 LMG 14566 LMG 14571 LMG 5829 LMG 1794T LMG 5916 LMG 14572 LMG 1244 LMG 14576 LMG 5822 LMG 5938 LMG 14573 LMG 14574 LMG 14575 LMG 5831 LMG 5939 LMG 5168 LMG 5167 LMG 14577 LMG5940 LMG 5833 LMG 14677 LMG 14675 LMG 2189 LMG 5848 LMG 7216 LMG 6812

Biovar

I I I I I I I I I I I I I I II III III III III III III III III IV IV V V V V V V nd nd nd nd

% Reassociation with labeled DNA from

Ribogroup

R38 R38 R38 R38 R38 R38 R38 R38 R38 R38 R38 R38 R42 R63 R59 R23 R23 R41 R44 R44 R44 R44 R64 R37 R46 R30 R36 R37 R37 R62 R62 R47 R61 R61 R65

LMG 1794 (bv. I)

LMG 14569 (bv. I)

41 48 47 40 45 43 nd 50 44 38 31 47 100 49 46 33 34 37 22 33 32 30 33 13 19 25 18 39 37 nd 40 34 40 39 32

100 82 81 72 72 70 70 66 66 66 66 63 42 47 38 32 28 32 32 33 28 29 32 30 19 34 14 48 48 47 39 30 44 44 48

(0) (0) (0) (2) (3) (4) (3) (3) (4) (5)

LMG 1244 (bv. III) 33 33 33 25 32 25 28 33 30 29 25 33 34 29 33 100 81 49 49 51 47 46 47 18 18 23 15 31 32 37 29 55 33 33 28

Abbreviations (Table 1-3): bv - Biovar nd - not determined LMG - Laboratorium voor Microbiologie Gent Culture Collection, Ghent, Belgium T - type-strain Numbers in brackets are ~Tm values in °C

nas sp.), R33 (P. asplenii), R45 (Pseudomonas sp.), R53d (P.stutzerii genomovar 3), and R75 (H.palleronii). In the following two ribogroups, the strains showed identical or almost identical patterns with SmaI and different patterns with HincH, R32 (P. fuscovaginae) and R66 (P. pertucinogena). In the following ribogroups, the strains showed identical patterns with HindI and different patterns with SmaI, R40 (P. agarici), R43, R54 (P. pseudoalcaligenes), R56 (P. alcaligenes), R57 (P. mendocina), and R78 (Brevundimonas diminuta). Thus, the combined numerical analysis was more precise than analysis of either SmaI or HindI data alone.

• Validity of computer analysis of ribotypes. In contrast to multilocus enzyme electrophoresis, which compares a single protein produced per each enzyme of a set (and thus can be used to trace a precise genealogy or phylogeny of strains), restriction fragment length polymorphism (including ribotyping) compares complex patterns. In the case of ribotyping, a band may be formed by one or several identical fragments originating from one or several copies of ribosomal RNA operons. Two strains showing a fragment of the same size may still differ by the origin of that fragment (165 or 235 gene). A single mutation can modify the size of a fragment and either generate a new band (case of a band formed by fragments originating

Ribotyping of Pseudomonas Table 2. DNA relatedness of Pseudomonas putida strains to the type strain

Present species P. P. P. P. P. P. P. P. P. P. P. P. P. P.

Table 3. DNA relatedness of environmental strains to the type strain of P. putida and a representative of the largest P. f/uorescens by. I group

putida putida putida putida putida putida putida putida putida putida putida putida putida putida

549

Strain designation

Ribogroup

Biovar

% Reassociation with labeled DNA from LMG 2257T

LMG2257T LMG 9070 LMG 2258 LMG 12644 LMG 2259 LMG 14679 t2 LMG5834 t1 LMG 2232 LMG 14680 LMG 14684 LMG 14683 LMG 14682 LMG 1246 t1 LMG 14681

R1 R1 R1 R1 R1 R1 R1 R7 R35 R35 R35 R35 R35 R35

A nd nd nd A A A nd B B B B B B

100 95 95 94 93 86 79 26 29 21 21 19 19 18

Present name

Strain designation

Ribogroup

Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp.

LMG 13977 LMG 13964 LMG 13959 LMG 13974 LMG 13980 LMG 13960 LMG 13972 LMG 13981 LMG 13971 LMG 13982 LMG 13988 LMG 13986 LMG 13961 LMG 13985 LMG 13983 LMG 13967

R1 R2 R4 R5 R8 Rll R20 R22 R28 R29 R34 R35 R37 R37 R43 R45

from different RNA operon copies) or move a band in the pattern. These modifications will result in one or two band differences. A single mutation can also join two fragments or split a fragment in two thus resulting in (1) two bands replaced by a slow-migrating band, (2) one band replaced by two fast-migrating bands, (3) addition of a slowmigrating band, or (4) addition of two fast migrating bands. Thus a single mutation can result in one to three band differences. Deletions or insertions can also occur complicating the interpretation of ribotypes. Thus, cluster analysis of ribotypes cannot be expected to give a reliable picture of phylogenie relationships among strains. However, when many ribotypes have to be studied, we need to compare all patterns in order to sort out those patterns which may be similar (epidemiological interest) or have several common bands suggesting a close relationship (even if the measure of relationship can be se-

% Reassociation with

P. putida 2257T

P. f/uorescens 14569 (bv I)

69 (5) 57 (6) 61 (7) 48 32 47 22 21 23 23 22 22 19 23 26 12

21 21 25 20 18 20 27 26 21 21 19 28 27 20 44 15

riously distorted). By using two endonucleases instead of one, the probability of occurrence of two unrelated strains with similar patterns is reduced. In addition to the abovementioned theoretical limitations, one must decide when two fragments have the same size in order to count similarity/dissimilarity values for a pair of patterns. Apparent size variation may be due to variation in electric field or temperature across the gel or to variation in DNA load. Many measurements made in ten years experience indicated that, for the gel format (4 standard lanes in a 20-lane gel) and standard (Citrobacter 32 DNA) used, fragment size variation is currently between 1 and 4%. Dendrograms obtained after distance values were calculated setting the tolerated fragment size variation to 3 or 5% showed only minor rearrangements of ribogroups (data not shown). The average clustering method used (Barthelemy and Guenoche, 1988) is a mod-

550

R. Brosch, M. Lefevre, F. Grimont, and P. A. D. Grimont

ification of UPGMA avoiding influence of strain order in the results. Although UPGMA gave virtually the same ribogroups, the order and respective position of ribogroups in the dendrogram were considerably affected by strain order (data not shown). • Aberrant strains. Strains P. chlororaphis LMG 5824, P. coronafaciens LMG 5081, P. putida by. B LMG 5837, and P. pseudoalcaligenes LMG 9369 which were found non authentic in other studies (Vancanneyt et al., 1996; Crimont et al., 1996) had aberrant positions in the ribotype dendrogram (ribotypes R77 and R72, respectively). These aberrant strains may be the result of mislabeling, substitution, or contamination at any stage of the strain history. Strains LMG 9369 (P. pseudoalcaligenes, R13), 10140 (P.chlororaphis, R1), and 2171 (P.putida, R48) showed a ribotype which differed from that of authentic strains although these strains could probably not be recognized as atypical without the help of modern methods. These strains were also found atypical (unclustered) in other studies (Vancanneyt et al., 1996; Crimont et al., 1996). These results demonstrate the ability of ribotyping to point out aberrant strains in a collection. • Ribogroups and bacterial genera. With the exception of ribogroups R69 and R72 which contained unauthentic strains (P. putida LMG 5837 and P. coronafaciens 5081, respectively), each ribogroup contained strains belonging to a single genus. However, the taxonomic hierarchy grouping species into genera was not clear in the ribotype dendrogram. Comamonas species clustered together and so did Acidovorax species. Comamonas spp., Acidovorax spp., and Hydrogenophaga palleronii formed a higher level cluster. Brevundimonas diminuta and H. pseudo{lava also formed a cluster. However, Burkholderia cepacia and Ralstonia solanacearum were located among Pseudomonas species.

P. aeruginosa, P. alcaligenes, P. stutzeri, P. pseudoalcaligenes, P. oleovorans, and P. mendocina strains formed a large cluster with a similar HincII pattern. Most of these species have clinical importance (Gavini et al., 1989). The common HincII-4 kpb band represents an interesting marker for strains of these species. It is noteworthy that this grouping is in good concordance with a scheme based on previous rRNAJDNA and DNAJDNA hybridization studies (Palleroni et al., 1972, 1973; Johnson and Pallero-

ni, 1989).

• Ribogroups and nomenspecies. Considering only those strains received with a species name and the ribogroups with at least two named strains, and after exclusion of three aberrant (misnamed or contaminant) strains, 38 of 41 (92.7%) ribogroups were homogeneous with respect to nomenspecies. Among the other ribogroups, one mixed P. chlororaphis and P. {luorescens (R30) and another (R61) mixed P. {luorescens and P. marginalis. Some nomenspecies were associated with more than one ribogroups. Strains of P. putida were found in ribogroups R1 and R35 (the strain in R69 was not authentic) and isolated ribotypes R3, R6, R7, R48. Strains of P. {luorescens were in ribogroups R23, R30, R37, R38, R44, R59, R61, and R62 and isolated ribotypes R21, R36, R41, R42, R46, R63, R64, and R65. P. marginalis strains were found in ri-

bogroups R19, R37, R43, R59, and R61. P. aeruginosa strains were in ribogroups R50 and R51. P. pseudoalcaligenes strains were in ribogroups R51, R54, and isolated ribotypes R13 and R58. P. alcaligenes strains were in ribogroups R52 and R56. The separation of P. aeruginosa by ribotyping was not very good. One strain (LMG 1272) showed three bands instead of two in the HindI pattern and a P. pseudoalcaligenes pattern (LMG 2854) clustered with P. aeruginosa LMG 1272 (R51). This might be a methodological artifact due to the equal weight given to all bands in pattern. Looking at the patterns in Fig. 1 (R51) shows that the pattern of P. pseudoalcaligenes LMG 2854 has some characteristics not shared by P. aeruginosa patterns. Strains formerly representing P. aureofaciens (LMG 1245, type strain, and LMG 5832) were found in ribogroup R30 with P. chlororaphis (formerly P. (luorescens by. D). Similarity of pulsed-field gel electrophoresis patterns of P. chlororaphis and P. aureofaciens had been observed (Crothues and Tummler, 1991). Following DNA-DNA hybridization studies, P. chlororaphis and P. aureofaciens were considered synonyms Uohnson and Palleroni, 1989). Bacterial species represented by a single strain constituted single ribotypes. These were P. taetrolens (R9), P. mucidolens (R27), P. synxantha (R39), R. solanacearum (R49), P. oleovorans (R55), and P. resinovorans (R67). • Ribogroups and biovars. Strains known to belong in P. putida by. A were found in ribogroups R1 and isolated ribotypes R3 and R6. Strains of P. putida by. B were in ribogroup R35 (a strain misnamed as P. putida by. B was also in ribogroup R69). Strains of P. {luorescens by. I were in ribogroup R38 and isolated ribotypes R21, R42 (type strain), and R63. The single strain of P. {luorescens by. II studied was in ribogroup R59 with P. marginalis strains. Historically, P. marginalis corresponded to plant-pathogenic strains of P. {luorescens by. II (Schroth et al., 1992). Strains of P. {luorescens by. III were in ribogroups R23 and R44 and isolated ribotypes R41 and R64. Strains of P. {luorescens by. IV were in ribogroups R37 and R46. Strains of P. {luorescens by. V were in ribogroups R37 and R62 and in the isolated ribotype R36. The following ribogroups contained strains of a single biovar (when known), R1 (P. putida by. A), R23 (P. (luorescens by. III), R35 (P. putida by. B), R38 (P. (luorescens by. I), R44 (P. (luorescens by. III), and R62 (P. (luorescens by. V). Thus, these P. {luorescens and P. putida biovars differ by ribotype in addition to phenotypic characters and may correspond to a taxonomic level above that of a biovar. • Ribogroups and pathovars. P. marginalis pv. marginalis were found in ribogroups R19, R37, R43, R59, and R61. P. marginalis pv. pastinacae and pv. alfalfae were in ribogroup R59. Ribogroup R19 contained only P. marginalis pv. marginalis and ribogroup R59 contained a mixture of the three different pathovars of P. marginalis. Thus, it is not clear whether ribotyping can retrieve pathovars. • Ribogroups and Biotype-100 phenons. Almost the same set of strains was studied by carbon source utilization tests using Biotype-100 strips (BioMerieux, La Balme-

Ribotyping of Pseudomonas les Grottes, France) and numerical taxonomy (Grimont et ai., 1996). Biotype-l00 phenons 1 to 34 are referred to as Biotype 1 to 34. An exclusive correspondence was observed in the case of R30-Biotype 7, R34-Biotype 5, R40Biotype 28, R45-Biotype 4, R47-Biotype 15, R53-Biotype 27, R66-Biotype 33, R69-Biotype 24, R72-Biotype 31, R73-Biotype 23, R75-Biotype 26, R78-Biotype 32, and R79-Biotype 22 (Table 4). The following ribogroups and Biotype phenons showed correspondence except for one strain, RI0-Biotype 9, R25-Biotype 14, R26-Biotype 17, and R50-Biotype 6. The following adjacent ribogroups corresponded to the same Biotype phenon, R32-R33Biotype 10 (except one strain), and R70-R71-Biotype 25. Strains in ribogroup R59 belonged to Biotype 10, 19, and 20 and all strains of Biotype 20 in that ribogroup had a 3.1 kb fragment. In addition, 10 of 13 strains which were not clustered into any Biotype phenon constituted unciustered ribotypes. • Ribogroups and Biolog phenons. Almost the same set of strains was studied by carbon source oxidation tests using Biolog GN Microplate system (Biolog, Hayward, CAl and numerical taxonomy (Grimont et aI., 1996). Biolog phenons 1 to 36 are referred to as Biolog 1 to 36. An exclusive correspondence was observed in the case of R8Biolog 21, R12-Biolog 27, R17-Biolog 28, R18-Biolog 26, R19-Biolog 16, R26-Biolog 5, R34-Biolog 22, R40-Biolog 23, R45-Biolog 13, R53-Biolog 24, R57-Biolog 20, R72Biolog 29, R75-Biolog 32, and R79-Biolog 36 (Table 4). The following ribogroups and Biolog phenons showed correspondence except for one strain, R15-Biolog 12, R25-Biolog 11, R50-Biolog 15, R73-Biolog 31, and R78Biolog 34. The following adjacent ribogroups corresponded to the same Biolog phenon, R69-R70-R71-Biolog 30, R28-R29-Biolog 14 (one exception), and R32-R33-Biolog 3 (one exception). In addition, 8 of 16 strains which did not cluster into any of the Biolog phenons constituted isolated ribotypes. • Ribogroups and DNA hybridization. It is currently agreed that a genomic species (genomospecies) is composed of strains closely related by DNA-DNA hybridization (at least 70% reassociation) with ~Tm values lower or equal to 5°C (Wayne et aI., 1987). Although percent relative reassociation is influenced by differences in genome size and by the method used, ~Tm values are more precise and independent from genome size differences or methods (Grimont, 1988). A good correlation was observed between DNA relatedness and position in a ribogroup (Table 1). Thus, the genomospecies P. putida was limited to ribogroup Rl. Other ribogroups containing strains labeled P. putida may represent other genomospecies. Ribogroup R38 constituted a genomospecies containing most strains of P. fluorescens bv. I. Five strains with DNA relatedness to LMG 14569 ranging from 63 to 66% were still considered to belong in that genomespecies since corresponding ~ Tm values were below or equal to 5 0c. ~ Tm is a stronger argument for or against assignment to a genomospecies since it is insensitive to genome size differences (Grimont, 1988). It is noteworthy that the type strain of P. fluorescens (also bv. I) corresponds to another genomospecies (with only one mem-

551

ber) and an isolated ribotype R42. Another culture of the type strain was received from Deutsche Sammlung fur Mikrobiologie (DSM 50090) and showed the same ribotype. In our laboratory, Louis Gardan had found the type strain of P. fluorescens to represent a genomospecies distinct from all other P. fluorescens strains studied (unpublished data). The subdivision of P. stutzeri into "genomovars" (Rossello et aI., 1991) correlated with the subdivision of ribogroup R53 in subgroups. Strains representing genomovar 1 (LMG 11199 and 2332) formed ribogroup R53b, strain LMG 5838 representing genomovar 2 was in ribogroup R53 c, and strains representing genomovar 3 (LMG 1228, 2334, and 6394) forming ribogroup R53d. According to the LMG catalog on the Internet, these latter three strains were derived from a single strain. Ribotyping may be used to sort out in a collection, those strains which may belong to different genomospecies. DNA-DNA hybridization is still necessary to check such hypothesis. • Elbe-river isolates. Ribotyping could suggest identification of 9 of the 30 environmental isolates tested with six taxons. According to their ribotypes, isolates LMG 13977 and LMG 13978 (Rl) were identified as P. putida biovar A. DNA relatedness of isolate 13977 with the type strain of P. putida with a ~ Tm value of 5°C confirmed the identification. Isolates LMG 13988 and 13986, clustered with P. fragi (R34) and P. putida bv. B (R35) strains, respectively. Isolates LMG 13985 and 13961 clustered with P. fluorescens bv. 4 and 5 (R37). Isolates LMG 12976 and 13975 formed a cluster (R71) very close to known representatives of C. testosteroni (R70). In fact, an analysis of phenotypic data clustered these strains with C. testosteroni (Grimont et aI., 1996). Isolate LMG 13983 clustered with a strain of P. marginalis (LMG 5170, R43), away from the type strain of that species. Thirteen other isolates formed 5 ribogroups not marked by the presence of a reference strain. Although ribogroups R2 and R4 clustered in the vicinity of P. putida bv. A strains, DNA relatedness and ~Tm values obtained with isolates LMG 13964 and 13959 confirmed their genomic differentiation from P. putida. Seven isolates gave isolated ribotypes. This identification exercise clearly indicates that many new species need to be described to allow identification of field isolates. A number of ribogroups formed by Elbe-river isolates probably represent new genomospecies. These results suggest the potential usefulness of ribotypes for species identification but also underline the need to establish a large database of ribotype for that purpose. In addition to species identification, ribotyping can indicate whether several isolates represent different strains. This may not be true for four Elbe-river isolates in ribogroup R2 and the isolates in ribogroup R5 and R45. Ribotyping may thus be used to select non redundant strains in a taxonomic study. • Comparison with other studies. Almost the same set of strains was submitted to SDS-PAGE (Vancanneyt et ai., 1996). The same aberrant strains were pointed out. Heterogeneity of P. fluorescens, P. marginalis, P. pseudoalcaligenes, P. putida, and P. stutzeri was also observed. Further-

R. Brosch, M. Lefevre, F. Grimont, and P. A. D. Grimont

552

Table 4. Ribogroups compared to clusters obtained by phenotypic analyses (Grimont et aI., 1996)

Ribo-

No."

Biolog

No." Biotype-IOO

phenon

group

R2

5

R3

I

No."

::

BLl9

20

BTI

20

R5

2

R6

1

R7

I

BLU

1

BTU

I

R8

2

BL21

2

BTll

2

R9

I

BLU

I

BTU

I

RIO

5

BL6

5

4 BTU I ................................................................................................................................ Rll I BLl9 I BT21 I

[BT9

Rl2

2

BL27

2

BT34

2

Rl3

I

BLU

I

BTU

I

Rl4

4

BL9

4

BTI3

4

Rl5

2

BLl2

2

BTI

2

Rl6

2

BLU

Rl7

3

BL28

Rl8

2

BL26

R19

2

BLl6

2

BTI2

R20

I

BLl

1

BTll

R21

I

BL2

1

BT9

R22

I

BLl

I

BTII

R23

2

BLl7

2

R24

4....

BL9

3

R25

5

BLlI

5

~~:

~~~

~··

R27

I

Biolog

No." Biotype-IOO

phenon

~]

BLl4

No."

phenon

3

BT3

3

................................................................................................................................

I

~~

No."

group

phenon

R4

.......

Ribo-

BLU

BLl8 3 ] 6.... [ 2 BT7 5 BLU ................................................................................................................................. R31 1 BLl2 I BT21 I R30

-::-~]~~u--:J~~~-----~-R34

R35

4

7

BL22

4

BT5

4

[:~:4

5

BTll

5

: ]

BIB

2

BLU

R36 I BLl I BTIO I ...................................................................................................••.......................•... R37

6

[:~:.

I

[

I

:

BL4 2 Brt6 2 .................................................................................................................................

= [:~u 13

7

~ :~

:]

8TI6

13

R39

I

BLU

I

BTU

I

2

R40

5

BL23

5

BT28

5

I

R41

I

BLl7

I

Brt8

I

R42

I

BL4

I

Brt6

I

1

R43

2

BL6

2

Brt9

2

BTI8

2

R44

5

BLl7

5

Brt8

5

BTI3

3

R45

2

BLl3

2

BT4

2

BTI4

5

R46

1

BL2

I

BT9

I

R47

6....

BLlO

4

Brt5

4

..·..[····i3Tli······················4······· BTU

1

BTU

I

[ BLl7 I Brt8 I .............................................................................................................•.................. R48 I BL U I BT 9 I

Ribotyping of Pseudomonas

553

Table4. Continued

Ribo-

No."

group

Biolog

No." Biotype-loo

phenon

R49

I

R50

6

R51

2

R52

No."

Ribo-

No."

group

phenon

Biolog phenon BL3S

2

BD3

2

R67

I

BLl9

I

BTU

I

I

R68

I

BLlI

I

BTI4

I

BDO

2

R69

BTI7

9

R70

7

BTI9

[BTI4 BTI5

4

R71

BTU

I

BLl5

7

BT6

7

BL33

I

BTI9

2

BL25

2

R53

9

BL24

9

R54

4

BL33

R55

1

BLU

R56

2

BL25

2

BDD

2

R57

3

BL2D

3

BTI

3

......R.H

R58

1

BL33

I

BTI9

I

R75

2

BL32

BL7

8

BTIO

8

BL4

8

BTI9

7

BL6

1

[ BTI6

2 R79

2

BL36

R59

18....

[

phenon

2

I

4

No."

R66

BLU

]

No." Biotype-loo

BTU

R60

I

R61

4

BL4

7

BTI6

7

R62

2

BLU

1

BTU

I

R63

I

R64

1

BLl7

I

BTI8

I

· · . R6S..· · ·. ·}"··. . . . ·..nL4·······················i···········titi6······················i·······

more, the characterization of Elbe-river isolates yielded the same results by SDS-PAGE and ribotyping. Ribotyping using endonuclease PvuII showed the heterogeneity of P. putida (Elomari et aI., 1994). Comparison of 16S rRNA sequences showed the type strain of P. f/uorescens to differ significantly from other P. f/uorescens strains, including representatives of different biovars (Moore et aI., 1996). • Taxonomic implications. Although the aim of the study was to evaluate the ability of ribotyping to uncover the taxonomic diversity of a complex bacterial group, results obtained with the collection of Pseudomonas (sensu lato) strains studied suggest some taxonomic action. The type strain (LMG 1794) of P. f/uorescens, although biochemically typical (Grimont et aI., 1996), is genomical-

.................................................................................................................................

3 4

R72

3

BL29

3

BD1

3

R73

~]

BL31

3

[BTI3

2

~!..~

~

2

BTI6

2

2

BTI2

2

.

* Number of strains characterized by the corresponding ribogroup, Biolog-c1uster, or Biotype-IOO cluster * * Not all strains from the corresponding ribogroup were also studied by Biolog/Biotype-IOO systems BL =Biolog phenon BT =Biotype-IOO phenon R = ribogroup U = unclustered strain

Iy too different from the commonly encountered P. f/uorescens strains. With molecular methods being introduced in

routine identification (through probes and RFLP), the risk is that the name P. f/uorescens disappears if limited to the present type strain (LMG 1794). Another type strain (preferably of biovar I) should be proposed for P. f/uorescens. As suggested by the results of this study, other P. f/uorescens biovars may represent new species. The species P. putida should include only strains of the present biovar A. Another species name should be given to strains of biovar B. Some isolated strains labeled P. putida by. A and showing unique ribotypes may represent new species. P. marginalis strains in different ribogroups should certainly be compared by DNA relatedness. From this work,

554

R. Brosch, M. Lefevre, F. Grimont, and P. A. D. Grimont

the subdivision of the species in different pathovars is not correlated with ribotyping data. The possible synonymy between P. asplenii and P. fuscovaginae was suggested by phenotypic studies (Goor, 1987; Grimont et aI., 1996) and SDS-PAGE (Vancanneyt et aI., 1996). These species which cause bacterial leaf blight of bird's nest firn (P. asplenii) and Oryza sativa (P. fuscovaginae) gave distinct ribotypes although several common fragments were observed (Fig. 1). DNA relatedness studies are required to demonstrate synonymy. It was interesting to observe the separation of P. caricapapayae from P. coronafaciens by ribotyping although phenotypic studies could not separate them (Grimont et aI., 1996). Although a few strains of P. syringae and related species were studied, a clear separation of P. syringae, P. ficuserectae, P. amygdali, P. meliae, and P. viridiflava was observed. Thus, ribotyping can be a useful taxonomic tool for phyto bacteriologists.

Conclusions This study showed clearly that the ribotype carries taxonomic information in addition to typing information. No other molecular typing method has been demonstrated to provide both identification and typing potentials. All studied species shown to be genomically valid species were separated by ribotyping using two endonucleases. A computer program, extracting fragment size data from ribotyping experiments, comparing patterns with a given tolerance on fragment size measurements, and cluster analysis, was the key element to uncover the taxonomic diversity of the studied organisms. Visual inspection of ribotypes on membranes can allow the detection of some identical patterns, but not the comparison of partially similar patterns when two endonucleases are used and many strains are studied. The next step in our methodological development will be to identify unknown isolates by automatic comparison of ribotypes against a ribotype database. The application of this ribotyping exercise to the genus Pseudomonas sensu lato raised a number of taxonomic hypotheses. These hypotheses should now be verified by DNA-DNA hybridization to allow genomospecies delineation. Genomospecies which can be differentiated by phenotypic characters will be eligible to receive Latin names (Wayne et aI., 1987) and such names facilitate international and intercultural communication. We learned from the HRAMI project that comparison of different methods for the characterization of bacteria is extremely fruitful. In practice, however, only a limited number of methods can be used. We recommend the use of a phenotypic method (we use Biotype-l00 strips) and a genomic method (we use ribotyping) for the identification of isolates when comprehensive databases are available. Sequencing of the 165 rRNA gene may be faster when the bacterial family (or phylogenie branch) to which the unknown isolate belongs is unknown. Identification ambiguities can often only be resolved by DNA relatedness studies.

Acknowledgements. This project was funded by CEC H program: "High-Resolution Automated Microbial Identification and Applications to Biotechnically Relevant Ecosystems" (contracts nr. BIOT-CT91-0294 and BI02-CT94-3098) of the European Commission. The authors thank M. Vancanneyt and K. Kersters from the University of Gent, Belgium, for supplying the strains used in this study. Our appreciation is extended to E. Ageron for helping initialize DNNDNA hybridization experiments.

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Patrick A. D. Grimont, Unite des Enterobacteries, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France