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 Aerobic bacteria Bacteria which require oxygen in order to grow and survive. Mentioned in: Aminoglycosides, Flesh-Eating Disease aerobic bacteria Bacteria that grow in the presence of O2 species associated with Galician oyster production was performed by the combination of numerical taxonomy Numerical taxonomy The grouping by numerical methods of taxonomic units based on their character states. The application of numerical methods to taxonomy, dating back to the rise of biometrics in the late nineteenth century, has received a great deal of 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 phytoplankton Flora of freely floating, often minute organisms that drift with water currents. Like land vegetation, phytoplankton uses carbon dioxide, releases oxygen, and converts minerals to a form animals can use. were compared with related sequences from the EMBL EMBL European Molecular Biology Laboratory EMBL Eniwetok Marine Biological Laboratory database. These results were used to identify the phenetic phe·net·ic adj. Of, relating to, or designating a system of classification of organisms based on overall or observable similarities rather than on phylogenetic or evolutionary relationships. 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 phe·no·type n. 1. a. The observable physical or biochemical characteristics of an organism, as determined by both genetic makeup and environmental influences. b. description of each identified species. It was mostly facultative facultative /fac·ul·ta·tive/ (fak´ul-ta?tiv) not obligatory; pertaining to the ability to adjust to particular circumstances or to assume a particular role. fac·ul·ta·tive adj. 1. psycrophilic [gamma]-Proteobacteria showing a great diversity. No specifity of bacteria, according to according to prep. 1. As stated or indicated by; on the authority of: according to historians. 2. In keeping with: according to instructions. 3. 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 pro·bi·ot·ic n. A dietary supplement containing live bacteria or yeast that supplements normal gastrointestinal flora, given especially after depletion of flora caused by infection or ingestion of an antibiotic drug. effect (Roseobacter gallaeciensis, Shewanella schlegeliana) or could be a potential risk for oyster cultures (Pseucloalteromonas piscicida, Pseudomonas anguilliseptica Pseudomonas anguilliseptica is a Gram-negative bacterium that is pathogenic to fish[1]. It was first isolated from Japanese eels (Anguilla japonica). Based on 16S rRNA analysis, P. anguilliseptica has been placed in the P. ) 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 bivalve, aquatic mollusk of the class Pelecypoda ("hatchet-foot") or Bivalvia, with a laterally compressed body and a shell consisting of two valves, or movable pieces, hinged by an elastic ligament. mollusc mollusc members of the phylum Mollusca, which comprises about 50,000 species. Includes snails, slugs and the aquatic molluscs—oysters, mussels, clams, cockles, arkshells, scallop, abalone, cuttlefish, squid. 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 Microbiota (human) Microbial flora harbored by normal, healthy individuals. A number of microorganisms have become adapted to a particular site or ecologic niche in or on their host. is essential for improving the industrial culture production, because it is the first step for designing a rapid presumptive pre·sump·tive adj. 1. Providing a reasonable basis for belief or acceptance. 2. Founded on probability or presumption. pre·sump guide for separating environmental and potentially pathogenic bacteria Pathogenic bacteria Bacteria that produce illness. Mentioned in: Gastroenteritis 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 larval 1. pertaining to larvae. 2. larvate. larval migrans see cutaneous and visceral larva migrans. , 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 UPGMA Unweighted Pair Group Method, Arithmetic Mean ) 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 phe·no·type n. 1. a. The observable physical or biochemical characteristics of an organism, as determined by both genetic makeup and environmental influences. b. 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 path·o·gen·ic or path·o·ge·net·ic adj. 1. Having the capability to cause disease. 2. Producing disease. 3. Relating to pathogenesis. 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 Agar, in the Bible Agar (ā`gər), the same as Hagar. agar, substance obtained from seaweed agar (ä`gär, ā`–, ăg`är) (Cultimed, Barcelona, Spain) and were stored at -80[degrees]C in Nutritive nutritive /nu·tri·tive/ (noo´tri-tiv) nutritional. nu·tri·tive adj. 1. Of or relating to nutrition. 2. Nutritious; nourishing. Broth broth liquid media for culturing microorganisms. cooked meat broth a medium useful for culturing anaerobic bacteria. enrichment broth one modified to permit growth by selected bacteria. (Cultimed, Barcelona, Spain) with 2% (w/v) NaCl (Pancreac, Barcelona, Spain) and 15% (v/v) of glycerol glycerol, glycerin, glycerine, or 1,2,3-propanetriol (prō`pāntrī'ŏl), CH2OHCHOHCH2OH, colorless, odorless, sweet-tasting, syrupy liquid. (Panreac). Sequencing of 16S rRNA Gene Sequencing of 23 strains (Table 1) representing all phena and some of unclustered strains of dendrogram A dendrogram is a tree diagram frequently used to illustrate the arrangement of the clusters produced by a clustering algorithm (see cluster analysis). Dendrograms are often used in computational biology to illustrate the clustering of genes. previously reported by us (Guisande et al. 2004) was performed according to the method described by Montes mon·tes n. Plural of mons. et al. (2003) using a 310 Genetic Analyzer (Applied Biosystems Applied Biosystems, Inc. (formerly NASDAQ: ABIO) is the original name of a pioneer biotechnology company founded in 1981 in Foster City, California, among the Silicon Valley cities of the southern San Francisco Bay Area. , Darmstadt, Germany) automated sequencer See MIDI sequencer. (music) sequencer - Any system for recording and/or playback of music via a programmable memory which stores music not as audio data, but as some representation of notes. . 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 DDBJ DNA Data Bank of Japan 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 phylogenetic tree Diagram showing the evolutionary interrelations of a group of organisms that usually originated from a shared ancestral form. The ancestor is in the tree trunk; organisms that have arisen from it are placed at the ends of tree branches. was constructed by using Molecular Evolutionary Genetics Evolutionary genetics is the broad field of studies that attempts to account for evolution in terms of changes in gene and genotype frequencies within populations and the processes that convert the variation with populations into more or less permanent variation between species. 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 See boot. (operating system, compiler) bootstrap - To load and initialise the operating system on a computer. Normally abbreviated to "boot". From the curious expression "to pull oneself up by one's bootstraps", one of the legendary feats of Baron von Munchhausen. 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 phy·lo·ge·net·ic adj. 1. Of or relating to phylogeny or phylogenetics. 2. Relating to or based on evolutionary development or history. 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 Pseudomonas A genus of gram-negative, nonsporeforming, rod-shaped bacteria. Motile species possess polar flagella. They are strictly aerobic, but some members do respire anaerobically in the presence of nitrate. (29 species), Pseudoalteromonas (24 species; it was abbreviated as Pa.) and Shewanella (33 species), and Proteobacteria-[alpha] subdivision with 3 species of Roseobacter. Nucleotide nucleotide (n  `klēətīd', ny`–), organic substance that serves as a monomer in forming nucleic acids. Sequence Accession Numbers Accession number may mean:
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 tryp·tic adj. Relating to or resulting from trypsin. tryptic relating to or resulting from digestion by trypsin. Soy Agar (TSA TSA See tax-sheltered annuity (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 A similarity matrix is a matrix of scores which express the similarity between two data points. Similarity matrices are strongly related to their counterparts, distance matrices and substitution matrices. 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 In statistics, and especially in biostatistics, cophenetic correlation (more precisely, the cophenetic correlation coefficient) is a measure of how faithfully a dendrogram preserves the pairwise distances between the original unmodeled data points. ) 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 CECT Contrast Enhanced Computed Tomography CECT Chemical Engineering and Chemical Technology (Coleccion Espanola de Cultivos Tipo, Valencia, Spain For the Valencia wine region, see . Valencia (Spanish: Valencia [ba'lenθja];[1] Valencian: València [va'ɫɛnsia]) is the capital of the Spanish autonomous community of Valencia and its province. ) [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 Marinobacter is a genus of Proteobacteria found in sea water. 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  The introduction of this article is too short.To comply with Wikipedia's lead section guidelines, it should be expanded. CECT [378.sup.T], Pseudomonas putida Pseudomonas putida is a gram-negative rod-shaped saprophytic soil bacterium. Based on 16S rRNA analysis, P. putida has been placed in the P. putida group, to which it lends its name[1]. 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 Escherichia coli (ĕsh'ərĭk`ēə kō`lī), common bacterium that normally inhabits the intestinal tracts of humans and animals, but can cause infection in other parts of the body, especially the urinary tract. 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 japonica (jəpŏn`əkə): see quince; camellia. were each included in one phenon, showing a high phenotypic homogeneity Homogeneity The degree to which items are similar. . All these aerobic strains were heterotrophic heterotrophic /het·ero·tro·phic/ (-tro´fik) not self-sustaining; said of microorganisms requiring a reduced form of carbon for energy and synthesis. 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 tax·o·nom·ic also tax·o·nom·i·cal adj. Of or relating to taxonomy: a taxonomic designation. tax 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 in·tra·spe·cif·ic also in·tra·spe·cies adj. Arising or occurring within a species: intraspecific competition. Adj. 1. 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 Pacific oyster n. An oyster (Crassostrea gigas) cultured in the United States and Europe, having a scalloped shell and a fruity flavor. Also called Portuguese oyster. (Crassostrea gigas), the genera genera, in taxonomy: see classification. 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 hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun) 1. crossbreeding; the act or process of producing hybrids. 2. molecular hybridization 3. with phylogenetic probes complementary to conserved regions The term Conserved region may refer to:
Water that makes up the oceans and seas. Seawater is a complex mixture of 96.5% water, 2.5% salts, and small amounts of other substances. Much of the world's magnesium is recovered from seawater, as are large quantities of bromine. , 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 larva, in zoology larva, independent, immature animal that undergoes a profound change, or metamorphosis, to assume the typical adult form. Larvae occur in almost all of the animal phyla; because most are tiny or microscopic, they are rarely seen. 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 Pathogen Any agent capable of causing disease. The term pathogen is usually restricted to living agents, which include viruses, rickettsia, bacteria, fungi, yeasts, protozoa, helminths, and certain insect larval stages. (Towner 1997, Levi & Rubinstein 1996), or Pa. tetraodonis that produce a tetrodotoxin tetrodotoxin /tet·ro·do·tox·in/ (tet´ro-do-tok?sin) a highly lethal neurotoxin present in numerous species of puffer fish and in certain newts (in which it is called tarichatoxin (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 col·o·nize v. col·o·nized, col·o·niz·ing, col·o·niz·es v.tr. 1. To form or establish a colony or colonies in. 2. To migrate to and settle in; occupy as a colony. 3. 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 PGIDIT02RMA (RealMedia Architecture) See RealMedia. 30102PR from the Xunta de Galicia The Xunta de Galicia is the political bureaucracy for the autonomous community of Galicia in Spain. 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In general, the input set of query sequences are assumed to have an evolutionary relationship by which they share a lineage and are descended from a 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 dinoflagellates minute aquatic protozoa; they produce red pigment and toxins which are taken up by shellfish without apparent ill effect, but the toxin is not metabolized and the shellfish may poison animals if eaten. : 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. 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Tamura, K. & M. Nei. 1993. Estimate of the number of nucleotide substitutions in the control region of mitochondrial DNA Mitochondrial DNA (mtDNA) is the DNA located in organelles called mitochondria. Most other DNA present in eukaryotic organisms is found in the cell nucleus. Nuclear and mitochondrial DNA are thought to be of separate evolutionary origin, with the mtDNA being derived from the 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 for·tis adj. Articulated with relatively strong pressure of the airstream below the glottis, as in English (p) and (t) compared with (b) and (d). n. A fortis consonant. 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 (PlayStation Portable) See PlayStation. 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 antibiotic resistance, n the ability of certain strains of microorganisms to develop resistance to antibiotics. antibiotic resistance of Acinetobacter spp Acinetobacter spp Bacteriology A widely distributed bacterium found in moist hospital environments, which may establish itself in the respiratory flora and on the skin of Pts with prolonged hospitalization, often via contaminated medical instruments–eg, . 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 vibrios (vib´rēōs´), n.pl bacteria belonging to the genus Vibrio found in plaque after 1 to 2 weeks of no flossing or brushing. 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 tidal flat Level muddy surface bordering an estuary, alternately submerged and exposed to the air by changing tidal levels. In addition to the alternating submergence and exposure, the varying influences of fresh river water and salty marine waters cause physical conditions 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 halophilic pertaining to or characterized by an affinity for salt; requiring a high concentration of salt for optimal growth. 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 Prado (prä`dō, Span. prä`thō), national Spanish museum of painting and sculpture, Madrid, one of the finest in Europe. , (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 Santiago de Compostela (säntyä`gō thā kōmpōstā`lä) or Santiago, city (1990 pop. 91,419), A Coruña prov., NW Spain, in Galicia, on the Sar River. , 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.
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