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An improved and rapid biochemical identification of indigenous aerobic culturable bacteria associated with Galician oyster production.

ABSTRACT An improved and rapid biochemical identification of the culturable aerobic bacteria species associated with Galician oyster production was performed by the combination of numerical taxonomy and sequencing of 16S rRNA gene. The 16S rRNA sequences of 23 representative bacterial isolates from different growing stages of Ostrea edulis, surrounding water, and phytoplankton were compared with related sequences from the EMBL database. These results were used to identify the phenetic clusters obtained by numerical taxonomy using the [S.sub.J]/UPGMA with a similarity level of 74%. The combination of the two techniques was a useful tool for identifying 40 out of 75 representative aerobic Gram negative isolates comprising the bacterial community studied and for improving the phenotypical description of each identified species. It was mostly facultative psycrophilic [gamma]-Proteobacteria showing a great diversity. No specifity of bacteria, according to the geographical area studied was found. Almost all of the identified species were associated, for the first time, with different growing stages of oyster. Some could have a probiotic effect (Roseobacter gallaeciensis, Shewanella schlegeliana) or could be a potential risk for oyster cultures (Pseucloalteromonas piscicida, Pseudomonas anguilliseptica) or for humans by consumption (Acinetobacter johnsonii, Pseudoalteromonas tetraodonis).

KEY WORDS: oyster, culturable aerobic bacteria, 16S rRNA gene, rapid identification

INTRODUCTION

The main culture of bivalve mollusc volume in Europe is concentrated in Galicia. Maintaining cultures with a high density increases the risks and consequences of infectious outbreaks. The study of natural culturable microbiota is essential for improving the industrial culture production, because it is the first step for designing a rapid presumptive guide for separating environmental and potentially pathogenic bacteria species. In a previous paper, we reported the phenotypical analyses of culturable aerobic bacteria associated with the Galician oyster (Ostrea edulis) cultures (Guisande et al. 2004). The samples were obtained monthly from oyster culture systems located on the Galician coast at: Bueu, Couso, Grove, Malpica, Ribadeo, and Vilagarcia de Arousa over 12 consecutive months, as was previously reported. A total of 397 isolates from different stages (seed, larval, and reproductive) of oyster, surrounding water and phytoplankton, selected as being representative of bacterial community, were characterized by numerical taxonomy. Nineteen per cent of isolates (75) were aerobic, gram negative, and using the Jaccard's coefficient at 69% ([S.sub.J]) and the unweighted pair group average method (UPGMA) a great diversity, and a high number of unidentified isolates were obtained.

The aim of our study was to select differential biochemical tests for a rapid identification of isolates after the identification by using the sequencing of 16S rRNA gene technique and numerical taxonomy analysis. A phenotypic description of each identified species will be provided. This study will differentiate between the strains associated with growing oyster stages, with a probiotic role or potentially pathogenic species for oysters or humans.

MATERIALS AND METHODS

Bacterial Strains

A total of 75 aerobic strains were obtained as representatives on a wide study of bacterial community associated with Galician oyster production from different stages (seed, larval, and reproductive) of oyster (Ostrea edulis), surrounding water, and phytoplankton (Guisande et al. 2004). Sampling, processing, and isolation of strains were previously reported by us. Pure cultures of strains were obtained on Marine Agar (Cultimed, Barcelona, Spain) and were stored at -80[degrees]C in Nutritive Broth (Cultimed, Barcelona, Spain) with 2% (w/v) NaCl (Pancreac, Barcelona, Spain) and 15% (v/v) of glycerol (Panreac).

Sequencing of 16S rRNA Gene

Sequencing of 23 strains (Table 1) representing all phena and some of unclustered strains of dendrogram previously reported by us (Guisande et al. 2004) was performed according to the method described by Montes et al. (2003) using a 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany) automated sequencer. The sequence of the 16S rRNA gene of strains was determined by using four primers (37F, 344F, 344R, and 1096R) and compared with sequences in public databases of GenBank, EMBL and DDBJ with BLAST, version 2.2.6. (Altschul et al. 1997). Multiple alignment of sequences was created by ClustalX, version 1.81 (Higgins & Sharp 1988), which included 1003 positions after removal of ambiguous positions (Hall 2001), using the BioEdit Sequence Alignment Editor, version 5.0.9 (Hall 1999). A phylogenetic tree was constructed by using Molecular Evolutionary Genetics Analysis (MEGA), version 2.1 (Kumar et al. 2001). This was performed using the neighbor-joining method (Saitou & Nei 1987) and Tamura-Nei distance model (Tamura & Nei 1993), with the calculation of cluster stability by bootstrap analysis with 1,000 replicates (Nei & Kumar 2000).

Sequences of Organisms Used for Phylogenetic Trees

All available species of each genus closest to studied strains were selected. In most cases, species type strains (if available) with nearly full-length 16S rRNA sequences were used. This includes members of two phylogenetic groups: Proteobacteria-[gamma] subdivision: Acinetobacter (16 species), Alteromonas (3 species; it was abbreviated as Alt.), Halomonas (27 species), Marinobacter (7 species), Mesorhizobium (5 species), Pseudomonas (29 species), Pseudoalteromonas (24 species; it was abbreviated as Pa.) and Shewanella (33 species), and Proteobacteria-[alpha] subdivision with 3 species of Roseobacter.

Nucleotide Sequence Accession Numbers

The partial 16S rRNA sequences of environmental isolates reported in this paper (42, 642, 29.98, 51.98, 73.98, 52.98, 55.98, 82.98, 251, 131, 173, 170, 174, 155, 86, 171, 111, 81, 117, 561, 8.98, 34.98, 38.98) have been deposited in the DDBJ (Mishima, Japan), EMBL (Heidelberg, Germany) and GenBank (Mountain View, USA) nucleotide sequence data bases under accession numbers AY870662 to AY870684, respectively.

Phenotypic Characterization

Bacterial strains and the reference strains were previously characterized by 92 physiological, morphological, and biochemical tests (Guisande et al. 2004). Cultures grown during 24 h at 22[degrees]C on Tryptic Soy Agar (TSA, Cultimed) and supplemented up to 2% (w/v) NaC1 (Panreac) (TSA 2%) were used as innocula. Data were processed with the NTSYS-pc, version 1.8 (Rohlf 1994). A similarity matrix was calculated using the [S.sub.J] at 74%. Phena were clustered using the UPGMA method and were defined with the earlier mentioned percentage. The correlation between the dendrogram and the similarity matrix (cophenetic correlation) was determined using the cophenetic correlation coefficient (r). The reproducibility of the tests was evaluated by analyzing 10% of strains in duplicate, as suggested by Sneath and Johnson (1972).

The reference strains included in the numerical taxonomy study were: Achromobacter denitrificans CECT (Coleccion Espanola de Cultivos Tipo, Valencia, Spain) [449.sup.T], Agrobacterium ferrugineum CECT [4356.sup.T], Halomonas aquamarina CECT [5000.sup.T], Marinomonas communis CECT [5003.sup.T], Marinomonas vaga CECT [5004.sup.T], Marinobacter hydrocarbonoclasticus CECT [5005.sup.T], Porphyrobacter sanguineus CECT [4271.sup.T], Pseudoalteromonas (abbreviated as Pa) citrea CECT [575.sup.T], Pa. espejiana CECT [5002.sup.T], Pa. haloplanktis CECT [4188.sup.T], Pa. undina CECT [5006.sup.T], Pseudomonas fluorescens CECT [378.sup.T], Pseudomonas putida CECT [324.sup.T], Shewanella hanedai CECT [5194.sup.T], and Stappia aggregata CECT [4269.sup.|T].

RESULTS

Sequencing of 16S rRNA Gene

The phylogenetic affiliation using the 16S rRNA sequence analysis was performed on 23 strains isolated from different growing stage of Ostrea edulis, surrounding water, and phytoplankton. They were representative within each phenon and some of them from unclustered strains of the previously reported dendrogram (Guisande et al. 2004). For most strains, sequences of the 16S rRNA gene, stretching from nucleotide positions 37-1040 (Escherichia coli equivalent), were obtained. These sequences were compared with each other and to related sequences, from the EMBL database, described in Material and Methods. The closest-neighboring species, which shared a similarity value in the 16S rRNA sequences of [greater than or equal to] 98%, were used to identify the isolates. This led to the identification of 20 strains from 23 selected strains, most of the identified strains belonging to the [gamma]-Proteobacteria. Only 3 were not identified, indicating species that have not been sequenced previously. So, the strains 8.98, 34.98, and 38.98 showed pair-wise sequence similarities of less than 95% to their nearest validly named neighbors (Fig. 1) and would probably correspond to new species.

[FIGURE 1 OMITTED]

The sequenced strains, geographical areas, and origin of identified species are shown in Table 1.

Phenotypic Characterization

The simplified dendrogram of aerobic strains showing the phena defined with a value of [S.sub.J] of 74% are presented in Figure 1. The average probability (P) of an erroneous test result (0.02) and the cophenetic correlation coefficient (r) (0.95) were acceptable values. The 60.44% (55/91) of strains were unclustered, and all the species identified by 16S rRNA sequence analysis were grouped into different phenetic clusters or were unclustered. Using the sequence of 16S rRNA gene 40 strains from 75 analyzed were identified. Pa. undina and P. mendocina included strains that were ungrouped. The strains identified as Alt. macleodii, M. flavimaris, and S. waksmanii were also grouped into separate phena (Fig. 1), confirming the presence of various biotypes in each of those species. The isolates identified as A. johnsonii, H. venusta, Pa. piscicida, and S. japonica were each included in one phenon, showing a high phenotypic homogeneity. All these aerobic strains were heterotrophic facultative psycrophilic, showing the same response for 49 tests (from the 87 analyzed), as previously described by Guisande et al. (2004). Characteristics not previously reported and differential tests of the identified species were shown in Table 2. From the 39 new reported tests, 35 were discriminatory to identify species. A selection of 4-15 tests for rapid identification of each species was made after developing a dichotomic differential table of species (Table 2).

DISCUSSION

The 16S rRNA sequence analysis has been used as a successful tool for identifying new strains (Stackebrant & Goebel 1994, Wiik et al. 1995, Patel et al. 1998, Farto et al. 2003, Montes et al. 2003) and for identifying indigenous bacteria population isolated from the natural environment (Gonzalez & Moran 1997, Kirchman 2002, Schauer et al. 2003). To clarify the taxonomic status of 75 representative aerobic isolates and select differential biochemical tests for a rapid identification of isolates from oyster culture, the phylogenetic affiliation using the 16S rRNA sequence analysis was performed on 23 strains. These strains were representative within each phenon, and some of them were from unclustered strains. The results showed that some of the isolates identified by sequencing as different species were grouped in the same phenetic cluster, making it difficult to obtain a reliable identification. To establish the phenetic clusters including only one species identified by 16S rRNA sequence analysis, we grouped the aerobic strains of our previous work (Guisande et al. 2004) by numerical taxonomy with a higher [S.sub.J] (74%) (Fig. 1). These results confirmed a high diversity of aerobic bacteria, as in Mediterranean oyster (Ostrea edulis) (Pujalte et al. 1999). The 16S rRNA sequence analysis led to the identification of 20 strains from 23 selected strains and 40 strains from 75 analyzed in this study (Table 2). Thus, both methods, numerical taxonomy and 16S rRNA sequences, were necessary for identification. The firs( one provided groups of strains with similar phenotypic characteristics (phenon) and the selection of representative strains within each phenon. The second method led to the molecular identification of each phenetic cluster by the selection of representative strains within each phenon. The combination of both techniques is a useful tool for identifying culturable isolates from unknown habitats with a great diversity. Most of the 16S rRNA sequencing data revealed the close phylogenetic relationship above the level proposed as the intraspecies variability ([greater than or equal to] 98%, Stackebrandt & Embley 2000), and the identification was performed according to the highest phylogenetic similarity value among the sequence of isolates and the reference strains. Moreover, the high number of bases obtained and the bootstrap analysis with 1,000 replicates were used for the identification because of the reliable results it gives and the positive outcome that it has had in phylogenetic studies (Ivanova et al. 2002, Hayashi et al. 2003, Satomi et al. 2003, Thompson et al. 2003, Yoon et al. 2003).

Among the more abundant microbiota associated with Canadian oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas), the genera Pseudomonas, Shewanella and Acinetobacter (Kueh & Chan 1985, Hariharan et al. 1995) were identified by phenotypical tests. We also found these genera in this work (Table 1), however, they were different from those associated with the Mediterranean oyster (Ostrea edulis), which were identified by hybridization with phylogenetic probes complementary to conserved regions of 16S rRNA (Pujalte et al. 1999). Unlike other studies, this study is the first research that includes the identification of culturable aerobic microbiota obtained from different stages of Ostrea edulis (seed, larval, and reproductive stage). In some cases, the same species was isolated from different stages of oyster and geographical area such as A. johnsonii, M. flavimaris, Pa. piscicida, Pa. undina, and S. waksmanii, confirming the non-specify of these species (Table 1). Among the species previously described as isolated from seawater, we have identified at different stages of oyster Aft. macleodii, M. flavimaris, Pa. undina, S. japonica, and S. pacifica (Gauthier et al. 1995, Ivanova et al. 2001, 2004. Yi et al. 2004, Yoon et al. 2004). Another species typically isolated from sediment, which we identified from oyster larva was S. livingstonensis (Bozal et al. 2002). Some other strains identified could be a potential risk for oyster cultures, because those species were associated with fish deaths, such as Pa. piscicida (Bein 1954), P. anguilliseptica (Domenech et al. 1997) or for human disease such as A. johnsonii, usually considered an opportunistic pathogen (Towner 1997, Levi & Rubinstein 1996), or Pa. tetraodonis that produce a tetrodotoxin (Simidu et al. 1990). We also identified species that could have a probiotic effect on oyster, such as Roseobacter gallaeciensis (Ruiz Ponte et al. 1999) and S. schlegeliana (Satomi et al. 2003), which were associated with a specific stage of oyster. Its potential use as probiotic should be tested. Finally, we also identified strains such as P. mendocina suggesting an earth contamination by soil (Satomi et al. 2003) or S. waksmanii showing the colonizing ability of different marine organisms, because other authors reported the association of this species with spincula (Ivanova et al. 2003). Thus, this is the first identification of these species associated with different growing stages of oyster.

Among the species isolated from seawater, we found A. johnsonii, Alt. macleodii, H. venusta, and Pa. piscicida. Other species of these genera were previously identified from seawater associated with the culture of Ostrea edulis in the Mediterranean by molecular methods (Pujalte et al. 1999). Alt. macleodii, H. venusta, and M. flavimaris were also isolated from phytoplankton. The genera Alteromonas and Marinobacter were also previously associated with phytoplankton by using molecular methods (Alavi et al. 2001, Hold et al. 2001, Seibold et al. 2001, Tobe et al. 2001, Green et al. 2004). The fact of identifying identical species on phytoplankton and oyster is probably a consequence of the process of filter feeding (Kueh & Chan 1985, Hariharan et al. 1995). Only H. venusta isolated from seawater and phytoplankton was excluded from oyster, suggesting the inability of this species to colonize this organism (Ostrea edulis).

Although there are a limited number of strains isolated from each source, it seems that there is more diversity associated with the larval than with the reproductive stage of oyster when comparing the number of identified species and the total number of identified strains (11/16 and 5/12, respectively; Table 1). However, we cannot evaluate the diversity with the seed, seawater, or phytoplankton, because the number of identified species or the total number of strains is too low (Table 1).

It is difficult to judge how numerically abundant or ecologically significant the species identified actually are, because their representation may simply reflect their selective enrichment in culture and not their numerical abundance. However, these isolates have a representative value as culturable aerobic bacteria associated with the culture of oyster in the NW of Spain because of the high number of strains isolated in different geographical areas over a long time period. The presence of various biotypes in Alt. macleodii, M. flavimaris, and S. waksmanii adds more phenotypical variability to each species (Farto et al. 1999, Farto et al. 2003, Montes et al. 2003), and this improves their phenotypical description.

A selection of phenotypical tests for rapid identification of potentially pathogenic or probiotic aerobic species was possible by the combination of both techniques. The improved and rapid identification of indigenous aerobic culturable bacteria could be extremely useful in industrial culture plants.

ACKNOWLEDGMENTS

The authors thank P. Moran for providing the automated sequencer. This work was supported by grant PGIDIT02RMA30102PR from the Xunta de Galicia (Regional Government of Galicia).

LITERATURE CITED

Alavi, M., T. Miller, K. Erlandson, R. Schneider & R. Belas. 2001. Bacterial community associated with Pfiesteria-like dinoflagellate culture. Environ. Microbiol. 3:380-396.

Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Z. Zheng Zhang, W. Miller & D. J. Lipman. 1997. Gapped BLAST & PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.

Bein, S. J. 1954. A study of certain chromogenic bacteria isolated from "red tide" water with a description of a new species. Bull. Mar. Sci. Gulf Caribb. 4:110-119.

Bozal, N., M. J. Montes, E. Tudela, F. Jimenez & J. Guinea. 2002. Shewanella frigidimarina and Shewanella livingstonensis sp. nov. isolated from Antarctic coastal areas. Int. J. Syst. Evol. Microbiol. 52: 195-205.

Domenech, A., J. F. Fernandez-Garayzabal, P. Lawson, J. A. Garcia, M. T. Cutuli, M. Blanco, A. Gibello, M. A. Moreno, M. D. Collins & L. Dominguez. 1997. Winter disease outbreak in sea-bream (Sparus aurata) associated with Pseudomonas anguilliseptica infection. Aquaculture 156:317-326.

Farto, R., M. Montes, M. J. Perez, T. P. Nieto, J. L. Larsen & K. Pedersen. 1999. Characterization by numerical taxonomy and ribotyping of Vibrio splendidus biovar I and Vibrio scophthalmi strains associated with turbot cultures. J. Appl. Microbiol. 86:796-804.

Farto, R., S. P. Armada, M. Montes, J. A. Guisande, M. J. Perez & T. P. Nieto. 2003. Vibrio lentus associated with wild diseased octopus (Octopus vulgaris). J. Invertebr. Pathol. 83:149-156.

Gauthier, G., M. Gauthier & R. Christen. 1995. Phylogenetic analysis of the genera Alteromonas, Shewanella and Moritella using genes coding for small-subunit rRNA sequences and division of the genus Alteromonas into two genera, Alteromonas (emended) and Pseudoalteromonas gen. nov., and proposal of twelve new species combinations. Int. J. Syst. Bacteriol. 45:755-761.

Gonzalez, J. M. & M. A. Moran. 1997. Numerical dominance of a group of marine bacteria in the [alpha]-subclass of the class Proteobacteria in coastal seawater. Appl. Environ. Microbiol. 63:4237-4242.

Green, D. H., L. E. Llewellyn, A. P. Negri, S. I. Blackburn & C. J. S. Bolch. 2004. Phylogenetic and functional diversity of the cultivable bacterial community associated with the paralytic shellfish poisoning dinoflagellate Gymnodinium catenatum. FEMS Microbiol. Ecol. 47: 345-357.

Guisande, J. A., M. Montes, R. Farto, S. P. Armada, M. J. Pdrez & T. P. Nieto. 2004. A set of tests for the phenotypic identification of culturable bacteria associated with Galician bivalve molluse production. J. Shellfish Res. 23:599-610.

Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41:95-98.

Hall, B. G. 2001. Phylognetic trees made easy: a how-to manual for molecular biologists. Sunderland, Massachusetts: Sinauer.

Hariharan, H., J. S. Giles, S. B. Heaney, G. Arsenault, N. Mcnair & D. J. Rainnie. 1995. Bacteriological studies on mussels and oysters from six river systems in Prince Edward Island, Canada. J. Shellfish Res. 14: 527-532.

Hayashi, K., J. Moriwaki, T. Sawabe, F. L. Thompson, J. Swings, N. Gudkovs, R. Christen & Y. Ezura. 2003. Vibrio superstes sp. nov., isolated from the gut of Australian abalones Haliotis laevigata and Haliotis rubra. Int. J. Syst. Evol. Microbiol. 53:1813-1817.

Higgins, D. G. & P. M. Sharp. 1988. CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73:237-244.

Hold, G. L., E. A. Smith, M. S. Rappe, E. W. Maas, E. R. B. Moore, C. Stroempl, J. R. Stephen, J. L. Prosser, T. H. Birkbeck & S. Galacher. 2001. Characterisation of bacterial communities associated with toxic and non-toxic dinoflagellates: Alexandrium spp. and Scrippsiella trochoidea. FEMS Microbiol. Ecol. 37:161-173.

Ivanova, E. P., T. Sawabe, N. M. Gorshkova, V. I. Svetashev, V. V. Mikhailov, D. V. Nicolau & R. Christen. 2001. Shewanella japonica sp. nov. Int. J. Syst. Evol. Microbiol. 51:1027-1033.

Ivanova, E. P., T. Sawabe, Y. V. Alexeeva, A. M. Lysenko, N. M. Gorshkova, K. Hayashi, N. V. Zukova, R. Christen & V. V. Mikhailov. 2002. Pseudoalteromonas issachenkonii sp. nov., a bacterium that degrades the thallus of the brown alga Fucus evanescens. Int. J. Syst. Evol. Microbiol. 52:229-234.

Ivanova, E. P., O. I. Nedashkovskaya, N. V. Zhukova, D. V. Nicolau, R. Christen & V. V. Mikhailov. 2003. Shewanella waksmanii sp. nov., isolated from a sipuncula (Phascolosoma japonicum). Int. J. Syst. Evol. Microbiol. 53:1471-1477.

Ivanova, E. P., N. M. Gorshkova, J. P. Bowman, A. M. Lysenko, N. V. Zhukova, A. F. Sergeev, V. V. Mikhailov & D. V. Nicolau. 2004. Shewanella pacifica sp. nov., a polyunsaturated fatty acid-producing bacterium isolated from sea water. Int. J. Syst. Evol. Microbiol. 54: 1083-1087.

Kirchman, D. L. 2002. The ecology of Cytophaga-Flavobacteria in aquatic environments. FEMS Microbiol. Ecol. 39:91-100.

Kueh, C. S. W. & K. Y. Chan. 1985. Bacteria in bivalve shellfish with special reference to the oyster. J. Appl. Bacteriol. 59:41-47.

Kumar, S., K. Tamura, I. B. Jakobsen & M. Nei. 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244-1245.

Levi, I. & E. Rubinstein. 1996. Acinetobacter infections overview of clinical features. Acinetobacter: microbiology, epidemiology, infections, management. In: I. Bergogne-Berezin, M. I. Joly-Guilloo & K. J. Towner, editors. Boca Raton: CRC Press. pp. 101-115.

Montes, M., R. Farto, M. J. Perez, T. P. Nieto, J. L. Larsen & H. Christensen. 2003. Characterization of Vibrio strains isolated from turbot (Scophthalmus maximus) culture by phenotypic analysis, ribotyping and 16S rRNA sequence comparison. J. Appl. Microbiol. 95:693-703.

Nei, M. & S. Kumar. 2000. Molecular evolution and phylogenetics. New York: Oxford University Press.

Patel, R., K. E. Piper, M. S. Rouse, J. M. Steckelberg, J. R. Uhl, P. Kohner, M. K. Hopkins, R. R. Cockerill, III & B. C. Kline. 1998. Determination of 16S rRNA sequences of Enterococci and application to species identification of non-motile Enterococcus gallinarum isolates. J. Clin. Microbiol. 36:3399-3407.

Pujalte, M. J., M. Ortigosa, M. C. Macian & E. Garay. 1999. Aerobic and facultative anaerobic heterotrophic bacteria associated to Mediterranean oysters and seawater. Int. Microbiol. 2:259-266.

Rohlf, F. J. 1994. Numerical Taxonomy and multivariate analysis system. Version 1.8. Department of Ecology and Evolution State University of New York. Exeter software.

Ruiz Ponte, C., J. F. Samain, J. L. Sanchez & J. L. Nicolas. 1999. The benefit of a Roseobacter species on the survival of scallop larvae. Mar. Biotechnol. 1:52-59.

Saitou, N. & M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.

Satomi, M., H. Oikawa & Y. Yano. 2003. Shewanella marinintestina sp. nov., Shewanella schlegeliana sp. nov. and Shewanella sairae sp. nov., novel eicosapentaenoic-acid producing marine bacteria isolated from sea animal intestines. Int. J. Syst. Evol. Microbiol. 53:491-499.

Schauer, M., V. Balague, C. Pedros-Alio & R. Massana. 2003. Seasonal changes in the taxonomic composition of bacterioplankton in a coastal oligotrophic system. Aquat. Microb. Ecol. 31:163-174.

Seibold, A., A. Wichels & C. Schutt. 2001. Diversity of endocytic bacteria in the dinoflagellate Noctiluca scintillans. Aquat. Microb. Ecol. 25: 229-235.

Simidu, U., K. Kita-Tsukamoto, T. Yasumoto & M. Yotsu. 1990. Taxonomy of four marine bacterial strains that produce tetrodotoxin. Int. J. Syst. Bacteriol. 40:331-336.

Sneath, P. H. A. & R. Johnson. 1972. The influence on numerical taxonomic similarities of errors in microbiological tests. J. Gen. Microbiol. 72:377-392.

Stackebrant, E. & B. M. Goebel. 1994. Taxonomic note: A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in Bacteriology. Int. J. Syst. Bacteriol. 44: 846-849.

Stackebrandt, E. & T. M. Embley. 2000. Diversity of uncultured microorganisms in the environment. Nonculturable microorganisms in the environment. In: R. R. Cowell & D. J. Grimes, editors. Washington DC: ASM. pp. 57-75.

Tamura, K. & M. Nei. 1993. Estimate of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10:512-516.

Thompson, F. L., C. C. Thompson, B. Hoste, K. Vandemeulebroecke, M. Gullian & J. Swings. 2003. Vibrio fortis sp. nov. and Vibrio hepatarius sp. nov., isolated from aquatic animals and the marine environment. Int. J. Syst. Evol. Microbiol. 53:1495-1501.

Tobe, K., C. Ferguson, M. Kelly, S. Gallacher & L. K. Medlin. 2001. Seasonal occurrence at a Scottish PSP monitoring site of purportedly toxic bacteria originally isolated from the toxic dinoflagellate genus Alexandrium. Eur. J. Phycol. 36:243-256.

Towner, K. J. 1997. Clinical importance and antibiotic resistance of Acinetobacter spp. J. Med. Microbiol. 46:721-746.

Wiik, R., E. Stackebrandt, O. Valle, F. L. Daae, O. M. Rodseth & K. Andersen. 1995. Classification of fish-pathogenic vibrios based on comparative 16S rRNA analysis. Int. J. Syst. Bacteriol. 45:421-428.

Yi, H., K. S. Bae & J. Chun. 2004. Aestuariibacter salexigens gen. nov., sp. nov. and Aestuariibacter halophilus sp. nov., isolated from tidal flat sediment, and emended description of Alteromonas macleodii. Int. J. Syst. Evol. Microbiol. 54:571-576.

Yoon, J. H., I. G. Kim, K. H. Kang, T. K. Oh & Y. H. Park. 2003. Alteromonas marina sp. nov., isolated from sea water of the east sea in Korea. Int. J. Syst. Evol. Microbiol. 53:1625-1630.

Yoon, J. H., S. H. Yeo, I. G. Kim & T. K. Oh. 2004. Marinobacter flavimaris sp. nov. and Marinobacter daepoensis sp. nov., slightly halophilic organisms isolated from sea water of the Yellow Sea in Korea. Int. J. Syst. Evol. Microbiol. 54:1799-1803.

ROSA FARTO, (1)* JOSE ANTONIO GUISANDE, (1) SUSANA P. ARMADA, (1) SUSANA PRADO, (2) AND TERESA P. NIETO (1)

* (1) Dpto. de Biologia Funcional y Ciencias de la Salud, Facultad de Biologia, Universidad de Vigo, Spain; (2) Dpto. de Microbiologia y Parasitologia, Universidad de Santiago de Compostela, Spain

* Corresponding author. E-mail: rfarto@uvigo.es
TABLE 1.
Identification and origin of aerobic bacteria species associated with
Galician oyster production on the basis of sequencing of the 16S rRNA
gene and numerical taxonomy.

 Origin of isolates

 Oyster (Ostrea edulis)

Species Seed Larval Reproductive

Acinetobacter
 A. johnsonii - + (31.98c, + (42#b)
 32.98c)
Alteromonas
 Alt. macleodii - + (17.98f, -
 622e, 642#e)
Halomonas
 H. venusta - - -
Marinobacter
 M. flavimaris + (14.98c) + (86.986, -
 73.98#f)
Pseudoalteromonas
 Pa. piscicida - + (61.98d) + (304.98b)
 Pa. tetraodonis - + (82.98#b) -
 Pa. undina + (251#c) + (131#b) -
Pseudomonas
 P. anguilliseptica - + (173#c) -
 P. mendocina - + (170#c, -
 174#c)
Roseobacter
 R. gallaeciensis + (155#c) - -
Shewanella
 S. japonica - - + (80b, 86#b, 87b)
 S. livingstonensis - + (171#c) -
 S. pacifica - + (111#b) -
 S. schelegeliana - - + (81#b)
 S. waksmanii - + (297.98b) + (117#b, 120b,
 123b, 549a,
 560c, 561#c)

 Origin of isolates

Species Seawater Phytoplankton

Acinetobacter
 A. johnsonii + (16.98f) -

Alteromonas
 Alt. macleodii + (29.98#f, 30.98f) + (50.98d)
Halomonas
 H. venusta + (57.98d, 59.98d) + (51.98#d)
Marinobacter
 M. flavimaris - + (52.98#d)
Pseudoalteromonas
 Pa. piscicida + (55.98#d) -
 Pa. tetraodonis - -
 Pa. undina - -
Pseudomonas
 P. anguilliseptica - -
 P. mendocina - -
Roseobacter
 R. gallaeciensis - -
Shewanella
 S. japonica - -
 S. livingstonensis - -
 S. pacifica - -
 S. schelegeliana - -
 S. waksmanii - -

In brackets: strain plus geographical area (a, Bueu; b, Couso;
c, Grove; d, Malpica; e, Ribadeo; f, Vilagarcia). In bold print:
selected strains for sequencing of 16S rRNA analysis.

Note: In bold print: selected strains for sequencing of 16S rRNA
analysis indicated with #.

TABLE 2.
Characteristics not previously reported and differential tests of
aerobic species identified

 Aerobic species identified *

Test 1 ([dagger]) 2 ([double
 dagger])

** 4 6
(1) ADH ([integral])([integral]) - -
Glucose oxidation (-) -
(1) KIA/[H.sub.2]S (-) -
Nitrate reduction v (-)
Growth at:
 4[degrees]C (+) - ([delta])
 10[degrees]C
 ([integral])([integral]) + (-)
 37[degrees]C (+) -
 44[degrees]C
 ([integral])([integral]) (-) -
 pH 10 + ([delta]) - ([delta])
Growth in:
 0.5% NaCl + (-)
 5% NaCl + +
 7% NaCl + +
 10% NaCl (-) v
 Crystal violet (-) (-)
 (1) TCBS Agar - -
 (1) TCBS (yellow) - -
Acid from:
 D-galactose (-) -
 D-mannose v -
Degradation of:
 Starch - +
 Esculin (-) +
Use as sole carbon source:
 Acetate + (+)
 [beta]-alanine + v
 DL-alanine + + ([delta])
 L-arginine + (+)
 Glycine + (+)
 Inulin + (-)
 L-lysine + (+)
 Malonate + (-)
 L-phenylalanine + + ([delta])
 L-proline + +
 Propanol + v
 Pyruvate + + ([delta])
 L-serine + (+)
 Succinate + v
 L-tartrate + ([delta]) (-)
 L-tryptophan + ([delta]) (-)
 Uracil + ([delta]) v
Sensitivity to:
 0/129 (150 [micro]g) - v
 Tetracycline (30 [micro]g)
 ([integral])([integral]) + v

 Aerobic species identified *

Test 3 ([dagger]) 4 ([double
 dagger])

** 3 4
(1) ADH ([integral])([integral]) - -
Glucose oxidation - -
(1) KIA/[H.sub.2]S - -
Nitrate reduction + ([delta]) + ([delta])
Growth at:
 4[degrees]C + ([delta]) - ([delta])
 10[degrees]C
 ([integral])([integral]) + v
 37[degrees]C + ([delta]) + ([delta])
 44[degrees]C
 ([integral])([integral]) - v
 pH 10 + ([delta]) + ([delta])
Growth in:
 0.5% NaCl + v
 5% NaCl + +
 7% NaCl + + ([delta])
 10% NaCl + ([delta]) (+)
 Crystal violet + ([delta]) (-)
 (1) TCBS Agar - -
 (1) TCBS (yellow) - -
Acid from:
 D-galactose - -
 D-mannose - -
Degradation of:
 Starch - (-)
 Esculin - (-)
Use as sole carbon source:
 Acetate + v
 [beta]-alanine v - ([delta])
 DL-alanine + ([delta]) -
 L-arginine + (-)
 Glycine v - ([delta])
 Inulin - -
 L-lysine + -
 Malonate + ([delta]) -
 L-phenylalanine - -
 L-proline + (+)
 Propanol v - ([delta])
 Pyruvate + v
 L-serine - - ([delta])
 Succinate + v
 L-tartrate - ([delta]) - ([delta])
 L-tryptophan - ([delta]) - ([delta])
 Uracil - ([delta]) - ([delta])
Sensitivity to:
 0/129 (150 [micro]g) nd nd
 Tetracycline (30 [micro]g)
 ([integral])([integral]) nd nd

 Aerobic species identified *

Test 5 ([dagger]) 6 ([parallel])

** 3 1
(1) ADH ([integral])([integral]) - -
Glucose oxidation - -
(1) KIA/[H.sub.2]S - -
Nitrate reduction - ([delta]) -
Growth at:
 4[degrees]C - ([delta]) + ([delta])
 10[degrees]C
 ([integral])([integral]) + +
 37[degrees]C - -
 44[degrees]C
 ([integral])([integral]) - -
 pH 10 + ([delta]) + ([delta])
Growth in:
 0.5% NaCl v -
 5% NaCl + +
 7% NaCl v +
 10% NaCl - -
 Crystal violet v -
 (1) TCBS Agar - -
 (1) TCBS (yellow) - -
Acid from:
 D-galactose nd nd
 D-mannose nd nd
Degradation of:
 Starch + +
 Esculin - nd
Use as sole carbon source:
 Acetate v -
 [beta]-alanine - -
 DL-alanine + ([delta]) - ([delta])
 L-arginine + -
 Glycine + -
 Inulin - -
 L-lysine - -
 Malonate - -
 L-phenylalanine v - ([delta])
 L-proline + ([delta]) -
 Propanol - ([delta]) -
 Pyruvate + ([delta]) -
 L-serine + ([delta]) -
 Succinate + -
 L-tartrate - ([delta]) -
 L-tryptophan - ([delta]) -
 Uracil - ([delta]) -
Sensitivity to:
 0/129 (150 [micro]g) + nd
 Tetracycline (30 [micro]g)
 ([integral])([integral]) + nd

 Aerobic species identified *

Test 7 ([integra]) 8 ([parallel])

** 2 1
(1) ADH ([integral])([integral]) - -
Glucose oxidation - -
(1) KIA/[H.sub.2]S - -
Nitrate reduction - ([delta]) + ([delta])
Growth at:
 4[degrees]C + ([delta]) + ([delta])
 10[degrees]C
 ([integral])([integral]) + +
 37[degrees]C - - ([delta])
 44[degrees]C
 ([integral])([integral]) - -
 pH 10 + ([delta]) + ([delta])
Growth in:
 0.5% NaCl - ([delta]) +
 5% NaCl v -
 7% NaCl v -
 10% NaCl v -
 Crystal violet v + ([delta])
 (1) TCBS Agar v -
 (1) TCBS (yellow) - ([delta]) -
Acid from:
 D-galactose nd + ([delta])
 D-mannose - -
Degradation of:
 Starch v -
 Esculin + -
Use as sole carbon source:
 Acetate v +
 [beta]-alanine - -
 DL-alanine + ([delta]) - ([delta])
 L-arginine - +
 Glycine - -
 Inulin - -
 L-lysine v -
 Malonate v - ([delta])
 L-phenylalanine + ([delta]) -
 L-proline + ([delta]) +
 Propanol + ([delta]) -
 Pyruvate + ([delta]) -
 L-serine + ([delta]) -
 Succinate + -
 L-tartrate - ([delta]) - ([delta])
 L-tryptophan - ([delta]) - ([delta])
 Uracil - ([delta]) - ([delta])
Sensitivity to:
 0/129 (150 [micro]g) v -
 Tetracycline (30 [micro]g)
 ([integral])([integral]) + +

 Aerobic species identified *

Test 9 ([integral] 10 ([parallel])

** 2 1
(1) ADH ([integral])([integral]) - -
Glucose oxidation - -
(1) KIA/[H.sub.2]S - -
Nitrate reduction + ([delta]) - ([delta])
Growth at:
 4[degrees]C - ([delta]) + ([delta])
 10[degrees]C
 ([integral])([integral]) + +
 37[degrees]C + ([delta]) -
 44[degrees]C
 ([integral])([integral]) - -
 pH 10 + ([delta]) + ([delta])
Growth in:
 0.5% NaCl + + ([delta])
 5% NaCl + +
 7% NaCl - ([delta]) -
 10% NaCl - -
 Crystal violet + -
 (1) TCBS Agar - + ([delta])
 (1) TCBS (yellow) - + ([delta])
Acid from:
 D-galactose - +
 D-mannose - -
Degradation of:
 Starch v +
 Esculin - +
Use as sole carbon source:
 Acetate + -
 [beta]-alanine + ([delta]) -
 DL-alanine v - ([delta])
 L-arginine v - ([delta])
 Glycine + ([delta]) -
 Inulin - -
 L-lysine v -
 Malonate v -
 L-phenylalanine v - ([delta])
 L-proline + - ([delta])
 Propanol + ([delta]) - ([delta])
 Pyruvate + - ([delta])
 L-serine + ([delta]) - ([delta])
 Succinate - -
 L-tartrate - ([delta]) - ([delta])
 L-tryptophan - ([delta]) - ([delta])
 Uracil - ([delta]) - ([delta])
Sensitivity to:
 0/129 (150 [micro]g) - nd
 Tetracycline (30 [micro]g)
 ([integral])([integral]) + nd

 Aerobic species identified *

Test 11 ([dagger]) 12 ([parallel])

** 3 1
(1) ADH ([integral])([integral]) - -
Glucose oxidation - ([delta]) - ([delta])
(1) KIA/[H.sub.2]S + ([delta]) - ([delta])
Nitrate reduction + ([delta]) + ([delta])
Growth at:
 4[degrees]C + ([delta]) + ([delta])
 10[degrees]C
 ([integral])([integral]) + +
 37[degrees]C - -
 44[degrees]C
 ([integral])([integral]) - -
 pH 10 + ([delta]) + ([delta])
Growth in:
 0.5% NaCl - + ([delta])
 5% NaCl v +
 7% NaCl - -
 10% NaCl - -
 Crystal violet - ([delta]) - ([delta])
 (1) TCBS Agar + -
 (1) TCBS (yellow) + ([delta]) -
Acid from:
 D-galactose - -
 D-mannose - -
Degradation of:
 Starch + ([delta]) -
 Esculin + +
Use as sole carbon source:
 Acetate - +
 [beta]-alanine - -
 DL-alanine - +
 L-arginine - -
 Glycine - -
 Inulin - -
 L-lysine - -
 Malonate - -
 L-phenylalanine - -
 L-proline - -
 Propanol - -
 Pyruvate - +
 L-serine - -
 Succinate - +
 L-tartrate - ([delta]) - ([delta])
 L-tryptophan - ([delta]) - ([delta])
 Uracil - ([delta]) - ([delta])
Sensitivity to:
 0/129 (150 [micro]g) + ([delta]) + ([delta])
 Tetracycline (30 [micro]g)
 ([integral])([integral]) + +

 Aerobic species identified *

Test 13 ([parallel]) 14 ([parallel])

** 1 1
(1) ADH ([integral])([integral]) - -
Glucose oxidation - ([delta]) - ([delta])
(1) KIA/[H.sub.2]S - - ([delta])
Nitrate reduction + ([delta]) + ([delta])
Growth at:
 4[degrees]C + ([delta]) + ([delta])
 10[degrees]C
 ([integral])([integral]) + +
 37[degrees]C - -
 44[degrees]C
 ([integral])([integral]) - -
 pH 10 + ([delta]) + ([delta])
Growth in:
 0.5% NaCl - - ([delta])
 5% NaCl + -
 7% NaCl - -
 10% NaCl - -
 Crystal violet - ([delta]) - ([delta])
 (1) TCBS Agar - -
 (1) TCBS (yellow) - -
Acid from:
 D-galactose - -
 D-mannose + -
Degradation of:
 Starch + -
 Esculin + -
Use as sole carbon source:
 Acetate - -
 [beta]-alanine - -
 DL-alanine - -
 L-arginine - -
 Glycine - -
 Inulin - -
 L-lysine - -
 Malonate - -
 L-phenylalanine - -
 L-proline - -
 Propanol - -
 Pyruvate - -
 L-serine - -
 Succinate - -
 L-tartrate - ([delta]) - ([delta])
 L-tryptophan - ([delta]) - ([delta])
 Uracil - ([delta]) - ([delta])
Sensitivity to:
 0/129 (150 [micro]g) + + ([delta])
 Tetracycline (30 [micro]g)
 ([integral])([integral]) + +

 Aerobic species identified *

Test 15 ([double dagger])

** 7
(1) ADH ([integral])([integral]) -
Glucose oxidation - ([delta])
(1) KIA/[H.sub.2]S v ([delta])
Nitrate reduction + ([delta])
Growth at:
 4[degrees]C (-) ([delta])
 10[degrees]C
 ([integral])([integral]) +
 37[degrees]C - ([delta])
 44[degrees]C
 ([integral])([integral]) -
 pH 10 + ([delta])
Growth in:
 0.5% NaCl - ([delta])
 5% NaCl (-)
 7% NaCl -
 10% NaCl -
 Crystal violet (+) ([delta])
 (1) TCBS Agar (+)
 (1) TCBS (yellow) - ([delta])
Acid from:
 D-galactose - ([delta])
 D-mannose -
Degradation of:
 Starch - ([delta])
 Esculin +
Use as sole carbon source:
 Acetate v
 [beta]-alanine (-)
 DL-alanine -
 L-arginine v
 Glycine (-)
 Inulin (-)
 L-lysine (-)
 Malonate -
 L-phenylalanine -
 L-proline (-)
 Propanol v
 Pyruvate v
 L-serine (-)
 Succinate v
 L-tartrate (-)
 L-tryptophan (-)
 Uracil - ([delta])
Sensitivity to:
 0/129 (150 [micro]g) - ([delta])
 Tetracycline (30 [micro]g)
 ([integral])([integral]) (+)

* 1, A. johnsonii; 2, Alt. macleodii; 3, H. venusta; 4, M. flavimaris;
5, Pa. piscicida; 6, Pa. tetraodonis; 7, Pa. undina; 8, P.
anguilliseptica; 9, P. mendocina; 10, R. galaeciensis; 11, S.
japonica; 12, S. livingstonensis; 13, S. pacifica; 14, S.
schelegeliana; 15, S. waksmanii.

([dagger]) All strains included in the same phena; ([double dagger])
strains included in differen phena; ([integra]) ungrouped strains;
([parallel]) one identified strain.

**, Number of identified strains of each species.

([delta] Useful discriminatory tests for rapid identification of each
species selected after making a dichotomic differential table of
species.

([integral)([integral]) No differential tests because all species gave
the same or variable result.

Data are expressed as: nd: No data; +: Positive result ([greater than
or equal to] 90% of positive results); -: Negative result ([greater
than or equal to] 10% of positive results); (+): Mainly positive
results ([greater than or equal to] 70% <90% of positive results);
(-): Mainly negative results ([greater than or equal to] 10% <30% of
positive results); v: Variable results (>30 <70% of positive results).

(1), ADH: Thornley's arginine dehydrolase; KIA: Kligler iron agar;
TCBS: thiosulphate citrate bile salt sucrose agar.
COPYRIGHT 2006 National Shellfisheries Association, Inc.
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Author:Nieto, Teresa P.
Publication:Journal of Shellfish Research
Date:Dec 1, 2006
Words:5826
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