INTRODUCTION

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Members of the genus Vibrio, of the family Vibrionaceae, have acquired increasing importance because several are associated with human disease. Vibrios of medical importance include Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio mimicus and, to a lesser extent, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, and Vibrio damsela (1, 2, 5, 7, 8, 13, 18). Recent studies report several clinical infections caused by Vibrio alginolyticus (7, 13, 18). Bacteremic infections occurred only in patients with underlying diseases (13). In the Mediterranean area, V. alginolyticus has been the cause of several ear infections in humans (18). Both biotypes of halophilous vibrios, Vibrio parahaemolyticus and V. alginolyticus, were responsible for intestinal diseases among the inhabitants of the littoral localities of the Crimea (21). In the United States (i.e., the gulf states Alabama, Florida, Texas, and Louisiana), raw oyster consumption is an important cause of Vibrio-associated gastroenteritis among adults without underlying illnesses (13).

Isolation of V. alginolyticus from Mediterranean coastal water has been reported in Spain (18) and in Sicily, Italy (6), although neither study genotypically characterized the strains isolated. Several methods have been reported to characterize different Vibrio species (1, 2, 3, 11, 12, 16, 20); outer membrane protein profiling and phage typing were the first molecular methods to be used (4, 14). Ribotyping has been applied successfully to differentiate various bacterial species, including V. vulnificus (1, 2, 11, 22). Recently, random amplified polymorphic DNA (RAPD) has been applied to differentiate Vibrio species (1, 2, 11, 12, 15, 20). RAPD is based on low-stringency amplification by decreasing the temperature of annealing (11, 15). The patterns obtained can vary greatly in response to minimal changes in the amplification. To interpret the patterns obtained, some studies group in the same cluster bacteria that have only 77% similarity (2); moreover, the reproducibility of the experiment is very low (15).

In order to characterize the V. alginolyticus strains isolated at the genotype level, we applied the ribotyping method and a new PCR fingerprinting method based on the amplification of conserved and specific internal sequences belonging to the IS256 family, which includes IS1245, IS1311, IS1081, and IS1395 (17).

	MATERIALS AND METHODS

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Bacterial strains. A total of 30 V. alginolyticus strains were isolated from samples of coastal water of northern Sardinia (Fig. 1). The samples were preenriched in alkaline peptone water (pH 8.6, 1% NaCl) at 37°C overnight, and yellow colonies were selected after 24 h of incubation on T.C.B.S. agar (Microbiol Diagnostici, Ca, Italy). The bacterial strains were presumptively identified by the API 20E system (BioMerieux, Marcy l'Etoile, France) (3). Ten were isolated from the Calich estuary of Alghero, three were from Porto Conte near Alghero, three were from the natural lake of Baratz, one was from a lake of Simbirizzi, seven were isolated in the seacoast of Tavolara island, and six were isolated from the seacoast waters of "La Maddalena" island. The strains were grown in marine broth 2216 (Microbiol Diagnostici) or Trypticase soy agar (Difco) supplemented with 0.5% (wt/vol) NaCl. Isolates were incubated at 25°C for 24 h.

View larger version (73K): [in this window] [in a new window]   FIG. 1.   Map of Sardinia. Arrows indicate the region of isolation of V. alginolyticus strains. 1, Tavolara; 2, La Maddalena; 3, Alghero; 4, Calich; 5, Baratz; 6, Simbirizzi.

DNA extraction and ribotyping. Chromosomal DNA extraction was performed as described by Wilson (25). The endonucleases HindIII, KpnI, and XbaI (Amersham International, Amersham, United Kingdom) were used to digest the chromosomal DNA in order to determine the best differentiation of V. alginolyticus strains as previously observed by Hoi et al. (11) and Arias et al. (1). HindIII was reported by Hoi et al. (11) to produce the best results, whereas we found XbaI to be the best enzyme for differentiating V. alginolyticus strains. Two micrograms of chromosomal DNA was digested with XbaI as specified by the manufacturer. To separate the DNA restriction fragments obtained, the fragments were added to electrophoresis gels in 0.8% agarose (FMC Bioproducts, Rockland, Maine) in 1× TAE (0.01 M Tris-acetate, 0.1 mM EDTA; pH 8) buffer at 35 V for 12 h. For hybridization, DNA was transferred to a nylon membrane (Amersham) by the Southern method (23). Hybridizations were carried out at 65°C, and the blots were washed at 68°C in 0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate; pH 7) and 0.1% sodium dodecyl sulfate. Probes were labeled by using the enhanced chemiluminescence gene detection system (Amersham) (20). The 1.8-kb ApaI clone (22) and  DNA were used as probes.

PCR fingerprinting. Primers were designed to be complementary to conserved regions of the 5' end of the IS1245 and IS1311 (10) and the 3' end of the IS1245 and 1311 belonging to the IS256 family (10, 17), and they were synthesized by using a Gene-Assembler Plus (Pharmacia LKB) in our institute. Sequences of the primers were 5'-TCACTACCGAGAGGAACATC-3' for p-95 and 5'-GGTGGAGGTGCCGTGCA-3' for p-447 and the primers were used at a concentration of 0.5 µM. Amplification reactions were performed in 50 µl with 1 U (final volume) of Taq polymerase, 20 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl, and 200 µM deoxynucleoside triphosphate (GIBCO-BRL/Life Technology, Paisley, United Kingdom). Reaction mixtures were overlaid with one drop of paraffin oil and then incubated for 2 min at 94°C, followed by 35 cycles at 94°C for 45 s, 58°C for 45 s, and 72°C for 1 min, with a final extension at 72°C for 20 min. The amplification products were visualized after electrophoresis at 90 V for 90 min in 2.5% Methaphore agarose gel (FMC Bioproducts), followed by staining of the gel with ethidium bromide. All DNA amplifications were performed in a Hybaid DNA thermal cycler instrument (Model TR3CM220; Omnigene).

Computer analysis of fingerprints. The patterns produced by the ribotyping method and PCR fingerprinting were evaluated with the Image Master software (Pharmacia). All bands produced were normalized by comparing the molecular weight markers (100-bp ladder) between different gels for PCR fingerprinting, and the molecular weights of the amplified bands were calculated by using the Image Master software. The dendrogram was generated by the UPGMA clustering method (deviation value of 0.025%).

	RESULTS

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Ribotyping. In Fig. 1 the regions where the 30 V. alginolyticus strains were isolated are shown. These strains were analyzed by ribotyping with the 3' part of the 16S, the intergenic spacer, and the 5' part of the 23S as a probe. The results are shown in Fig. 2 and Table 1. We used three restriction enzymes, HindIII, KpnI, and XbaI, in order to obtain the best differentiation of the V. alginolyticus strains. XbaI produced seven different patterns of the 30 strains analyzed, whereas KpnI and HindIII only produced four and three patterns, respectively (data not shown). The 10 strains isolated in the region of Calich, on the west coast of Sardinia, showed three different profiles (Table 1; Fig. 2, lanes A and D, and a profile similar to that shown in lane C). All three strains from Alghero generated ribotype pattern 6, as did one strain from La Maddalena and one strain from Tavolara (Fig. 2, lane C). The 13 strains from the La Maddalena and Tavolara islands generated three patterns of hybridization (types 1, 2, and 6) with the prevalence of pattern 1 (Table 1). In Fig. 2, lane G, is shown the pattern of strain L15 (type 1, isolated in La Maddalena sea waters); the other patterns found (type 2 and 6) are shown in Fig. 2, lanes B and C. These two islands are both on the Sardinian east coast and they are very close geographically, as can be seen in Fig. 1. The three strains isolated in the salty lake of Baratz showed the same ribotype (type 7; Fig. 2, lane E), whereas the Simbirizzi isolate generated a unique pattern (type 5; Fig. 2, lane F).

View larger version (172K): [in this window] [in a new window]   FIG. 2.   Southern blot of chromosomal DNA showing representative examples of the different patterns obtained by ribotyping of the analyzed V. alginolyticus isolates. Lanes: A, L5 (Calich); B, L6 (Tavolara); C, L28 (Alghero); D, L29 (Calich); E, L7 (Baratz); F, L27 (Simbirizzi); G, L15 (La Maddalena); and MW,  HindIII marker.

PCR fingerprinting. On the basis of conserved sequences among bacteria, we chose two primers complementary to conserved sequences of the transposase gene of IS1311 and IS1245 (10, 17). Representative examples of the results obtained are shown in Fig. 3, where we can observe the relatively large number of bands amplified from the different isolates (bands 2 to 6). In order to test the reproducibility of the technique, we amplified the chromosomal DNA of the different isolates after several subcultures; all of the strains tested showed the same amplification patterns in the different experiments performed (data not shown). In total, PCR fingerprinting of the 30 isolated V. alginolyticus strains produced 12 different profiles (Table 1). The patterns obtained were evaluated by the Image Master system analysis in order to determine the molecular weights of the bands, and the genetic distances among these strains were evaluated by the construction of a dendrogram, prepared with the Dendron software, on the basis of the patterns obtained (Fig. 4). The PCR fingerprinting clearly shows a higher level of differentiation than did the ribotyping. In particular, the three ribotyping families produced by the 13 strains from the La Maddalena and Tavolara islands were subdivided into six subfamilies by the PCR fingerprinting method (Table 1 and Fig. 4). The 10 strains isolated in the Calich region of Alghero, which generated three ribotype patterns, showed five different patterns of amplification, whereas the three strains from the lake of Baratz confirmed the results obtained with ribotyping, since they generated the same pattern of amplification (Table 1 and Fig. 3). The isolate from the artificial lake of Simbirizzi, L27, which showed a unique ribotyping pattern (type 5), also produced a unique pattern with PCR fingerprinting (Table 1 and Fig. 4).

View larger version (113K): [in this window] [in a new window]   FIG. 3.   Agarose gel electrophoresis of DNA amplifications of V. alginolyticus isolates obtained with the PCR fingerprinting method. Lanes: MW, 100-bp ladder (GIBCO/Life Science); 1, L1 (Calich); 2, L2 (Calich); 3, L3 (Calich); 4, L4 (Calich); 5, L5 (Calich); 6, L6 (Tavolara); 7, L7 (Baratz); 8, L8 (Baratz); 9, L9 (Baratz); 10, L10 (La Maddalena); 11, L11 (Tavolara); 12, L12 (Tavolara); 13, L13 (Tavolara); 14, L14 (Alghero); 15, L15 (La Maddalena); 16, L16 (Tavolara); 17, L17 (La Maddalena); 18, L18 (La Maddalena); 19, L19 (Calich); 20, L20 (Alghero); 21, L21 (Calich); 22, L22 (Calich); 23, L23 (La Maddalena); 24, L24 (La Maddalena); 25, L25 (Tavolara); 26, L26 (Tavolara); 27, L27 (Cagliari); 28, L28 (Alghero); and 29, L29 (Calich).

View larger version (33K): [in this window] [in a new window]   FIG. 4.   Dendrogram with restriction fragment length polymorphism profiles illustrating the relationships among the 30 V. alginolyticus strains analyzed by PCR fingerprinting. Increasing similarity resulted in similarity index (SAB) values ranging from 0 to 1.0.

	DISCUSSION

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Several studies have been reported that use different strategies to fingerprint V. vulnificus strains (1, 2, 3, 11, 20). Among the molecular techniques used, ribotyping is the most diffuse (1, 2, 3, 11). Recently, the introduction of RAPD PCRs has shortened the time of typing (15). Enterobacterial repetitive consensus sequence PCR has been reported to produce polymorphic patterns when applied to different Vibrio species (9), but some studies claim that the complex patterns generated are produced by a mechanism similar to the RAPD method (9).

We report here the characterization of 30 V. alginolyticus strains isolated from Sardinian waters by ribotyping and a new PCR fingerprinting based on the amplification of specific insertion sequences. Ribotyping generates fingerprinting patterns reproducible over time, but it is not able to detect minimal changes in the bacterial genome, nor can it detect virulence characters which can change more rapidly than ribotype patterns within a bacterial species. In these cases we need a method that can detect such changes. In fact, although the patterns that we obtained from ribotyping were reproducible and easy to interpret, this method produced only seven different patterns from 30 different bacterial strains. The PCR method showed a greater ability to differentiate the strains analyzed, producing 12 different patterns. The strains isolated in the La Maddalena and Tavolara islands and those isolated in Alghero produced only three patterns by ribotype (types 1, 2, and 6; the latter type was found also in the Alghero and Calich isolates), whereas PCR fingerprinting could differentiate the same strains into six different families. The PCR fingerprinting method proposed is based on the use of primer sequences that are complementary to conserved IS sequences (10, 24). This PCR method was able to differentiate within ribotype families. It is possible that the amplified products obtained were not necessarily generated by annealing to genuine IS256 sequences in the target genome, but they could be generated by amplification of other related insertion sequences. The observation that the PCR banding patterns obtained with these primer pairs were quite similar at annealing temperatures of both 50 and 60°C indicates that primer annealing is largely sequence specific rather than random. This PCR fingerprinting method is rapid and simple, and it may be a helpful tool for differentiating V. alginolyticus strains in epidemiological analyses, particularly in large studies and in urgent situations.