A set of tests for the phenotypic identification of culturable bacteria associated with Galician bivalve mollusc production.ABSTRACT To select a set of biochemical tests that provide a rapid identification of culturable bacteria associated with Galician 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. molluscs cultures, the phenotypic diversity of some bacterial species was investigated. A total of 488 strains isolated from water, 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. , 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. , seed and reproductive phases in oysters and clams cultures in Galicia (NW of Spain) and 51 type and reference strains were characterized by 92 physiologic, morphologic and biochemical tests. The 80% of isolates were facultative anaerobes and the remaining 20% were aerobes. Using 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 probabilistic methods, 57% of facultative anaerobic anaerobic /an·aer·o·bic/ (an?ah-ro´bik) 1. lacking molecular oxygen. 2. growing, living, or occurring in the absence of molecular oxygen; pertaining to an anaerobe. isolates could be identified: Vibrio vibrio Any of a group of aquatic, comma-shaped bacteria in the family Vibrionaceae. Some species cause serious diseases in humans and other animals. They are gram-negative (see aestuarianus, V. splendidus biotype biotype /bio·type/ (bi´o-tip) 1. a group of individuals having the same genotype. 2. any of a number of strains of a species of microorganisms having differentiable physiologic characteristics. I, V. alginolyticus, V. mytili. V. (Listonella) anguillarum, V. salmonicida. V. furnissii and V. orientalis. The predominant aerobic strains were Shewanella spp., Pseudoalteromonas spp,. 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. spp. and Alcaligenes denitrificans. Vibrio was the most frequently isolated genus from oysters, clams and their culture water. Pseudomonas and Pseudoalteromonas were the main isolated genus from phytoplankton samples. A selection of a set of tests for the phenotypic and presumptive pre·sump·tive adj. 1. Providing a reasonable basis for belief or acceptance. 2. Founded on probability or presumption. pre·sump identification of the main culturable bacterial groups was possible. Since this set of tests provides a rapid identification, it will be very useful in the quality control of bivalve mollusks, in industrial treatment plants or in the work of hygiene and health control staff. KEY WORDS: oysters, clams, presumptive bacterial identification, 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 , facultative anaerobic bacteria Anaerobic bacteria Bacteria that do not require oxgyen, found in low concentrations in the normal vagina Mentioned in: Aminoglycosides, Bacterial Vaginosis, Flesh-Eating Disease, Periodontal Disease , Vibrio, numerical taxonomy INTRODUCTION The culture of bivalve molluscs represents the 8% of the world fish production and the 26% of the world aquaculture aquaculture, the raising and harvesting of fresh- and saltwater plants and animals. The most economically important form of aquaculture is fish farming, an industry that accounts for an ever increasing share of world fisheries production. production. Bivalve mollusk mollusk: see Mollusca. mollusk or mollusc Any of some 75,000 species of soft-bodied invertebrate animals (phylum Mollusca), many of which are wholly or partly enclosed in a calcium carbonate shell secreted by the mantle, a soft production has been increasing in the last 50 years worldwide, with a triplication TRIPLICATION, pleading. This was formerly used in pleading instead of rebutter. 1 Bro. Civ. Law, 469, n. of its production in the last decade (Lovatelli 2003). Spain is the main bivalve mollusks producer in Europe, with an annual production of 275,000 tons, where the main culture volume is concentrated in Galicia (NW of Spain). The development of modern techniques allows the maintenance of cultures with a high density of mollusks, making intensive cultures possible but also increasing the risks and consequences of infectious outbreaks. The association of microorganisms with animal tissues can be symbiotic symbiotic /sym·bi·ot·ic/ (sim?bi-ot´ik) associated in symbiosis; living together. sym·bi·ot·ic adj. Of, resembling, or relating to symbiosis. or pathogenic or, as in the case of mollusks, coincidental co·in·ci·den·tal adj. 1. Occurring as or resulting from coincidence. 2. Happening or existing at the same time. co·in with the process of filter feeding. The great phenotypic diversity of these bacterial strains makes identification of isolates difficult. Also, certain bacterial species have been described as pathogenic to bivalve mollusks and/or to people who consume them. These species include: V. tapetis (Paillard pail·lard n. A slice of veal, chicken, or beef that is pounded until very thin and cooked quickly. [Origin unknown.] et al. 1989, Castro et al. 1993, Castro 1994, Robledo et al. 1994, Borrego et al. 1996), V. tubiashii (Tubiash et al. 1965), V. vulnificus (Tracket et al. 1984, Horre & Gunter 1996), V. (Listonella) anguillarum (Grischkowsky & Liston 1974, Elston 1989, Montilla et al. 1994), V. fluvialis (Lee et al. 1981), V. pelagius (Montilla et al. 1994), V. mimicus (Matte et al. 1994), Vibrio cholerae Vibrio chol·er·ae n. A bacterium that causes Asiatic cholera in humans; Koch's bacillus. Vibrio cholerae Infectious disease The Vibrio (Kaper et al. 1983), V. parahaemolyticus (Kaper et al. 1983), V. alginolyticus (Grischkowsky & Liston 1974, Elston 1989), or Plesiomonas shigelloides Plesiomonas shigelloides Aeromonas shigelloides, Pseudomonas shigelloides Bacteriology A bacterium of brackish or salt water, which may cause gastroenteritis due to exposure to contaminated water or seafood (Prescott et al. 1999). The aim of this study is to identify the bacteria associated with the culture of bivalve molluscs in Galicia to select tests that can be useful to separate environmental bacteria species associated with mollusks cultures and the most frequent potentially pathogenic strains. Such a set of tests will be very useful for undertaking the quality control of bivalve mollusks, in industrial treatment plants or in the work of hygiene and health control staff. MATERIALS AND METHODS Bacterial Strains Samples were obtained from different stages (seed, larval larval 1. pertaining to larvae. 2. larvate. larval migrans see cutaneous and visceral larva migrans. , and reproductive) in cultures of clams (Ruditapes decussatus, Venerupis pullastra, Tapes japonica japonica (jəpŏn`əkə): see quince; camellia. ) and oysters (Ostrea edalis), as well as from the surrounding water and phytoplankton. All animals were washed first to remove loosely associated bacteria and contaminants. All sterilizations were done at 121[degrees]C for 15 min and all filtrations through 0.22-[micro]m pore size porosity filters. Reproductive bivalve mollusks (50 g of meat and intervalval liquid) were homogenized ho·mog·e·nize v. ho·mog·e·nized, ho·mog·e·niz·ing, ho·mog·e·niz·es v.tr. 1. To make homogeneous. 2. a. To reduce to particles and disperse throughout a fluid. b. in 100 mL of sterile marine phosphate-buffered saline (PBS PBS in full Public Broadcasting Service Private, nonprofit U.S. corporation of public television stations. PBS provides its member stations, which are supported by public funds and private contributions rather than by commercials, with educational, cultural, ) (20 g of NaCl per liter, 0.2 g of KCl per liter, 1.15 g of P[O.sub.4]H[Na.sub.2] per liter, 0.2 g of P[O.sub.4][H.sub.2]K per liter; pH 7.1 to 7.2). Bivalve mollusks in the larval and seed stages (50 units each) were separately homogenized in 100 mL of PBS. Phytoplankton samples were obtained through the filtration of 1000 mL of water and their suspension in 100 mL of PBS. Serial dilutions (1:10; 1:100; 1:1000) of all these samples were plated in duplicate 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) (MA, Cultimed, Barcelona, Spain) and Thiosulphate-Citrate-Bile-Sucrose (TCBS TCBS Tea Club and Barrovian Society (from Tolkien novel) TCBS The Bear Creek School TCBS Trunked Common Base Station , Cultimed), including water samples. Plates were incubated at 22[degrees]C for 4 days. Five colonies of each morphology were picked off and striked on MA to obtain pure cultures, which were inoculated on Nutritive nutritive /nu·tri·tive/ (noo´tri-tiv) nutritional. nu·tri·tive adj. 1. Of or relating to nutrition. 2. Nutritious; nourishing. Broth (Cultimed) with 2% (w/v) NaCl (Panreac, 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) for their conservation at -80[degrees]C. Sampling was carried out over 12 consecutive months (1 sample a month) in bivalve mollusks cullure systems located on the Galician coasts at: Bueu, O Grove O Grove (Galician) , Ogrobe (Galician alternative) or El Grueve (Spanish) is a municipality in Galicia, Spain, belonging to the province of Pontevedra. [ edit ] Municipalities in Pontevedra prep. 1. As stated or indicated by; on the authority of: according to historians. 2. In keeping with: according to instructions. 3. these methods, 480 strains were isolated. The following 51 type and reference strains were included in this study for comparative purposes and were characterized under the same conditions and using the same tests as for the remainder of the isolates: Agrobaeterium ferrugineum CECT CECT Contrast Enhanced Computed Tomography CECT Chemical Engineering and Chemical Technology (Coleccion Espanola de Cultivos Tipo, Valencia, Spaiu) 4356, Ag. sanguineum CECT [4271.sup.T], Ag. stellulatum CECT 4269, Alcaligenes denitrificans CECT [449.sup.T], Halomonas aquamarina CECT [5000.sup.T], Marinomonas communis CECT [5003.sup.T], M. vaga CECT [5004.sup.T], Photobacterium angustum CECT [4193.sup.T], Ph. damselae subsp. damselae ATCC ATCC American Type Culture Collection, see there (American Type Culture Collection American Type Culture Collection (ATCC) is a private, not-for-profit biological resource center whose mission focuses on the acquisition, authentication, production, preservation, development and distribution of standard reference microorganisms, cell lines and other materials for , Manassas. VA, USA) [33539.sup.T], Ph. damselae subsp. piscicida ATCC 17911, Ph. phosphoreum CECT 4172, Pseudoalteromonas citrea CECT [575.sup.T], Pseudoalteromonas espejiana CECT [5002.sup.T], Pseudoalteromonas haloplanktis Pseudoalteromonas haloplanktis is a marine bacterium. CECT [4188.sup.T], Pseudoalteromonas 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], Ps. nautica CECT [5005.sup.T], Ps. putida CECT [324.sup.T], Shewanella hanedai CECT [5194.sup.T], Vibrio aestuarianus ATCC [35048.sup.T], V. albensis LMG LMG Light Machine Gun LMG Laurence M. Gould (Antarctic Research Support Vessel, USAP) LMG Local Marketing Group LMG Loaf's Merry Guild LMG Laboratory Molecular Genetics LMG Liquid Methane Gas (Laboratorie vor Microbiologic, Universiteit Gent, Belgium) 4406 (V. cholerae, Garrity et al. 2002), V. alginolyticus CECT [521.sup.T], V. anguillarum 43-F, NCIMB NCIMB National Collection of Industrial Food and Marine Bacteria (UK) (Nationals Collections of Industrial and Marine Bacteria, Aberdeen, UK) 571 aud NCIMB 6 (Listonella anguillarum Listonella anguillarum is a Gram-negative marine bacteria in the family Vibrionaceae. . Garrity et al. 2002), V. campbellii CECT [523.sup.T], V. carchariae LMG [7890.sup.T] (V. harveyi. Garrity et al. 2002), V. costicola LMG 6460 (Salinivibrio costicola subsp, costicola, Garrity et al. 2002), V. diazotrophicus LMG [7893.sup.T], V. fischeri LMG [4414.sup.T], V. fluvialis LMG [7984.sup.T], V. furnissii LMG [7910.sup.T], V. harveyi LMG [4044.sup.T], V. lentus CECT 5110, V. metschnikovii ATCC 7708, V. mimicus LMG [7896.sup.T], V. mytili CECT p[632.sup.T], V. natriegens LMG [10935.sup.T], V. nereis ne·re·is n. pl. ne·re·i·des See clamworm. [Latin N  r LMG
[3895.sup.T], V. nigripulchritudo CECT [628.sup.T], V. ordalii CECT
[582.sup.T], V. orientalis CECT [629.sup.T], V. parahaemolyticus LMG
[2850.sup.T], V. pelagius CECT [4202.sup.T] (Listonella pelagia, Garrity
et al. 2002), V. prateolyticus LMG [3772.sup.T], V. salmonicida ATCC
[43839.sup.T], V. scophthalmi AO[89.sup.T]. V. splendidus biotype I ATCC
[33125.sup.T], V. tapetis CECT [4600.sup.T], V. tubiashii LMG
[10936.sup.T], and V. vulnificus biotype II CECT 897.
Phenotypic Characterization Bacterial strains were characterized by 92 physiologic, morphologic, and biochemical tests. 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) NaCl (TSA 2%) were used as inocula. Unless otherwise indicated, all incubations were undertaken at 22[degrees]C, commercial media were supplemented up to 2% (w/v) NaCl and results were read after 48 hours. All tests and methods were carried out as described by Montes mon·tes n. Plural of mons. et al. (1999), except that the hydrolysis hydrolysis (hīdrŏl`ĭsĭs), chemical reaction of a compound with water, usually resulting in the formation of one or more new compounds. of urea was tested on Stuart's Urea Broth (Pronadisa, Madrid, Spain) and the Gram character was performed using the KOH KOH The chemical formula for potassium hydroxide, which is used to perform the KOH test. The tests is also called a potassium hydroxide preparation. Mentioned in: KOH Test KOH potassium hydroxide. 3% (v/v) (Analema, Vigo, Spain) method (Buck 1982). All tests were assayed on plates and were spot inoculated with a multipoint inoculator, except for O/F O/F On File O/F Overflow (oxidative or fermentative fer·men·ta·tive adj. 1. Causing or having the ability to cause fermentation. 2. Relating to or of the nature of fermentation. metabolism of glucose), ODC ODC - Open Distributed Computing (ornithine decarboxylase The enzyme ornithine decarboxylase (ODC) is a homodimer of 461 amino acids (in humans, at least). Reaction It catalyzes the decarboxylation of ornithine producing, as a result, diamine putrescine: ), LDC LDC See: Less developed countries LDC See less developed country (LDC). (lysine decarboxylase Lysine decarboxylase is an enzyme which converts lysine to cadaverine. External links
• • ), HDC (Hard Disk Controller) See disk controller. HDC - Disk Controller (histidine decarboxylase histidine decarboxylase n. An enzyme that catalyzes the decarboxylation of histidine to histamine. ), ADH ADH: see antidiuretic hormone. (arginine arginine (är`jənĭn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer participates in the biosynthesis of proteins. dihydrolase), Voges-Proskauer, methyl red Methyl Red, also called C.I. Acid Red 2, is an indicator dye that turns red in acidic solutions. It is an azo dye, and is a dark red crystalline powder. Methyl red is a pH indicator; it is red in pH under 4.4, yellow in pH over 6.2, and orange in between. , Kligler, nitrate reduction, indole indole /in·dole/ (in´dol) a compound obtained from coal tar and indigo and produced by decomposition of tryptophan in the intestine, where it contributes to the peculiar odor of feces. It is excreted in the urine in the form of indican. production, and the hydrolysis of urea when tests were performed in tubes. Coding of Data Results were coded using the following criteria: 1 for positive results, 0 for negative results and 9 for non-comparable or missing values In statistics, missing values are a common occurrence. Several statistical methods have been developed to deal with this problem. Missing values mean that no data value is stored for the variable in the current observation. . Data were processed with the NTSYS-pc, version 1.8 (Rohlf 1994). Similarity matrices were calculated using the Simple Matching coefficient ([S.sub.SM]) and Jaccard's coefficient ([S.sub.J]. Phena were clustered using the Unweighted Pair Group Method, with Arithmetic mean (mathematics) arithmetic mean - The mean of a list of N numbers calculated by dividing their sum by N. The arithmetic mean is appropriate for sets of numbers that are added together or that form an arithmetic series. (UPGMA UPGMA Unweighted Pair Group Method, Arithmetic Mean ). The correlation between the 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. and each 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. (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 Sheath and Johnson (1972). For phena that could not be identified by numerical taxonomy methods, probabilistic (probability) probabilistic - Relating to, or governed by, probability. The behaviour of a probabilistic system cannot be predicted exactly but the probability of certain behaviours is known. Such systems may be simulated using pseudorandom numbers. identification was performed with the "Probabilistic identification of bacteria" program, version 1.10 (Bryant 1995). This computer program includes the data matrices of Bryant et al. (1986) and Alsina and Blanch blanch to become pale. (1994a), providing the most suitable probabilistic identification of each phenon. RESULTS Numerical Taxonomy Seventy-six per cent of a total of 488 strains were isolated in MA and the remaining 24% in TCBS. Some 80% of isolates were Gram negative, oxidase oxidase /ox·i·dase/ (ok´si-das) any enzyme of the class of oxidoreductases in which molecular oxygen is the hydrogen acceptor. ox·i·dase n. positive and with the ability to ferment ferment /fer·ment/ (fer-ment´) to undergo fermentation; used for the decomposition of carbohydrates. fer·ment n. 1. glucose (facultative anacrobic strains). The remaining 20% were Gram negative, oxidase positive, and non-fermentative strains (aerobic strains). The probability of an erroneous result was P = 2.596% for aerobic strains and P = 2.045% for facultative anaerobic strains. According to the criteria suggested by Sheath and Johnson (1972), each of these values and their average (2.3078%) are acceptable values (lower than 5%) and are indicative of good reproducibility. Five tests were deleted from the data matrices because of their variances (higher than 0.1, according to Bryant et al. 1986): acid production from arbutine, use of aspartate aspartate /as·par·tate/ (ah-spahr´tat) a salt of aspartic acid, or aspartic acid in dissociated form. a·spar·tate n. 1. A salt of aspartic acid. 2. or alginate alginate /al·gi·nate/ (al´ji-nat) a salt of alginic acid; water-soluble alginates are useful as materials for dental impressions. as sole carbon source, production of lipase lipase (lī`pās), any enzyme capable of degrading lipid molecules. The bulk of dietary lipids are a class called triacylglycerols and are attacked by lipases to yield simple fatty acids and glycerol, molecules which can permeate the membranes using tween tween n. A child between middle childhood and adolesence, usually between 8 and 12 years old. [Blend of teen1 and between.] 20 as substrate and [beta]-hemolysis. Furthermore, 9 tests giving the same result were also eliminated for aerobic strains: growth at 22[degrees]C (+: positive result), growth at pH 7.5 (+), oxidase (+), histidine histidine (hĭs`tĭdēn), organic compound, one of the 22 α-amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein. decarboxylation de·car·box·yl·a·tion n. Removal of a carboxyl group from a chemical compound, usually with hydrogen replacing it. decarboxylation (dē´karbok´s (-: negative result), glucose fermentation (-), Voges Proskauer test (-), acid production from inositol inositol (ĭnō`sĭtōl): see vitamin. Inositol The generic name for hexahydroxycyclohexanes, which are classified as carbohydrates. (-) or L-rhamnose (-), and the Gram character, that was negative. For facultative anaerobic strains, 9 tests were deleted from the data matrix, as they had always shown the same result: growth at 22[degrees]C (+), growth at pH 4.5 (-), oxidase (+), glucose fermentation (+), glucose on Kligler (+), acid production from inositol (-) or raffinose Raffinose The best-known trisaccharide (oligosaccharide), widely distributed in higher plants. The best-known sources are cottonseed meal and the manna of Eucalyptus. (-), motility motility /mo·til·i·ty/ (mo-til´ite) the ability to move spontaneously.mo´tile Motility Motility is spontaneous movement. (+) and the Gram character, that was negative. For the aerobic strains, the number of final matrices was 106 (strains) x 78 (tests) and for facultative anaerobic strains the number was 434 (strains) x 78 (tests). Similarities were calculated using the [S.sub.SM] and [S.sub.J] coefficients. Phena were clustered using the UPGMA. Cophenetic correlation showed the best fit (Rohlf 1994) using the [S.sub.J] coefficient (r = 0.81621 for aerobic strains and r = 0.80555 for facultative anaerobic strains). This study focuses on clustered isolates: 57% of aerobic strains (with a [S.sub.j]/UPGMA similarity level of 69%) and 88% of facultative anaerobic strains (with a [S.sub.J]/UPGMA similarity level of 69%). Aerobic Strains A phenotypic dendrogram constructed using the [S.sub.J]/UPGMA analysis included 23 phena (Fig. 1) with a similarity level of 69%, clustering 57% of aerobic isolates. Only 4 isolates were identified: Shewanella hanedai (one isolate), Alcaligenes denitrificans (one isolate), Pseudoalteromonas espejiana (one isolate) and Pseudomonas nautica (one isolate). [FIGURE 1 OMITTED] Independently of the [S.sub.J] similarity level and due to the great diversity of unidentified aerobic isolates that was found, five different main groups were defined (see Fig. 1) taking shared characteristics of close phena into account: Shewanella spp. (16% of clustered isolates), low-NaCl requirements for growth (16% of isolates), Pseudoalteromonas spp. (15% of isolates), Pseudomonas spp. (8% of isolates), and Alcaligenes denitrificans (1% of isolates). In addition to the tests for which the same result was obtained, as described earlier for all aerobic strains, clustered aerobic isolates always displayed the same response for the following tests: growth at 28[degrees]C (+), pH 4.5 (-), pH 9 (+), growth in 3.5% (w/v) NaCl (+), [H.sub.2]S production on TCBS (-), catalase reaction catalase reaction (kat´  lās),n the response of bubbling in the presence of hydrogen peroxide given by blood exudates or transudates. (+), ODC and LDC (-); oxidative character on O/F medium (+), utilization of glucose or lactose on Kligler medium (-), methyl red reaction (-), indole production (-), [alpha]-haemolysis (-), acid production from amygdaline amygdaline /amyg·da·line/ (-len?) 1. like an almond. 2. tonsillar. a·myg·da·line adj. 1. Relating to or resembling an almond. 2. , L-arabinose, D-cellobiose, ethanol, D-fructose, Dgalactose, glycerol, lactose, D-mannitol, D-melibiose, D-raffinose, ribose, sucrose, salicia, sorbilol or D-trehalose (-); degradation of urea, chitin, gelatin gelatin or animal jelly, foodstuff obtained from connective tissue (found in hoofs, bones, tendons, ligaments, and cartilage) of vertebrate animals by the action of boiling water or dilute acid. , casein casein (kā`sēn), well-defined group of proteins found in milk, constituting about 80% of the proteins in cow's milk, but only 40% in human milk. , DNA DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. , lecithin lecithin Any of a class of phospholipids (also called phosphatidyl cholines) important in cell structure and metabolism. They are composed of phosphate, choline, glycerol (as the ester), and two fatty acids. Various fatty acids pairs distinguish the various lecithins. , chondroitin chondroitin (k  n·droiˑ·tin),n , cellulose or agar (-); utilization of citrate citrate /cit·rate/ (sit´rat) a salt of citric acid. citrate phosphate dextrose (CPD) anticoagulant citrate phosphate dextrose solution. as sole carbon source (-), and motility (+). Differential phenotypic characteristics are given in Table 1. The main characteristics that allow different groups to be defined are cited below: Shewanella spp. This group was constituted by phenon 1 (two isolates), phenon 2 (two isolates), phenon 3 (four isolates), phenon 4 (three isolates), phenon 5 (one isolate and the reference strain of Shewanella hanedai), and phenon 6 (three isolates). All showed a mesophilic character (growth between 10[degrees]C and 28[degrees]C) and had NaCl requirements for growing, despite being unable to tolerate high NaCl concentrations (7% or even 5% in some cases). Half of them were able to produce [H.sub.2]S on Kligler medium, and most grew on TCBS medium and carry out nitrate reduction, as well as growth maintenance at pH 10 and sensitivity to tetracycline tetracycline (tĕ'trəsī`klēn), any of a group of antibiotics produced by bacteria of the genus Streptomyces. They are effective against a wide range of Gram positive and Gram negative bacteria, interfering with protein (30 [micro]g/disc). They were generally unable to use any of the assayed substrates as sole carbon source. Low-NaCl Requirements for Growth This group included phena 8 (7 isolates), 10 (4 isolates), 12 (2 isolates), and 13 (2 isolates). The main characteristics of this group are growth maintenance in low NaCl concentrations (0.5%), which indicates the low NaCl requirements for growth, as well as NaCl tolerance of up to 7%, growth maintenance at pH 10 and the use of different substrates as sole carbon source: acetate, [beta]-alanine, DL-alanine, L-arginine, L-lysine, malonate mal´o`nate a. 1. (Chem.) A salt of malonic acid. malonate A salt or ester of malonic acid. , L-proline, propanol pro·pa·nol n. See propyl alcohol. , pyruvate pyruvate /py·ru·vate/ (pi´roo-vat) a salt, ester, or anion of pyruvic acid. Pyruvate is the end product of glycolysis and may be metabolized to lactate or to acetyl CoA. py·ru·vate n. , L- serine serine (sĕr`ēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein. or succinate succinate /suc·ci·nate/ (suk´si-nat) any salt or ester of succinic acid. succinate semialdehyde ?. suc·ci·nate n. . All had sensitivity to tetracycline (30 [micro]g/disc). Pseudoalteromonas spp. This group included the following phena: 7 (1 isolate and the reference strain of Pseudoalteromonas espejiana), 16 (2 isolates), 17 (3 isolates), 18 (3 isolates), 19 (3 isolates), and 20 (2 isolates). A reference strain of Pseudoalteromonas haloplanktis was clustered close to phenon 20. All these strains showed NaCl require merits for growth, tolerating most of them NaCl concentrations of up to 7%. These strains were able to grow at 28[degrees]C (but not usually at 37[degrees]C) and to carry out starch degradation. Glycine glycine (glī`sēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Glycine is the only one of these amino acids that is not optically active, i.e. and L-proline were the sole substrates they could use as carbon sources. Pseudomonas spp. This group was constituted by phenon 15 (one isolate was clustered with reference strains of 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]. and Pseudomonas fluorescens), phenon 21 (2 isolates), phenon 22 (2 isolates were clustered with a reference strain of Pseudomonas nautica), and phenon 23 (2 isolates). Most of them were able to grow between 10[degrees]C and 28[degrees]C (even at 44[degrees]C in some cases), at pH 10 and had low-NaCl requirements for growth (0.5% NaCl). They were able to use acetate, L-proline and pyruvate as sole carbon sources and were sensitive to tetracycline (30 [micro]g/disc). Alcaligenes denitrificans One bacterial isolate was clustered with the reference strain of Alcaligenes denitrificans (phenon 9). Both were able to grow between 10[degrees]C and 37[degrees]C and at pH 10; showed low-NaCl requirements for growth and were not able to tolerate high NaCl concentrations (7% or even 5% in some cases). They were able to carry out nitrate reduction and the use of different substrates as sole carbon source, such as acetate, [beta]-alanine, DL-alanine, glycine, L-lysine, malonate, L-phenylalanine, L-proline, propanol, pyruvate, or L-serine. They were sensitive to tetracycline (30 [micro]g/disc), but not to O/129 (150 [micro]g/disc). The following 6 tests (Table 1) are proposed as discriminatory and useful for the biochemical identification of Gram negative, motile mo·tile adj. 1. Moving or having the power to move spontaneously. 2. Of or relating to mental imagery that arises primarily from sensations of bodily movement and position rather than from visual or auditory sensations. , oxidase positive, aerobic, and able to grow at 22[degrees]C strains associated with the culture of Galician bivalve mollusks: growth in different concentrations of NaCl (0.5% and 7%), nitrate reduction, growth on TCBS medium and the use of DL-alanine or malonate as sole carbon source. Facultative Anaerobic Strains A dendrogram was obtained applying Jaccard's similarity coefficient and using the UPGMA as a clustering method. In total, 41 phena (clustering 88% of facultative anaerobic strains) were established with a [S.sub.J]/UPGMA similarity level of 69% (Fig. 2). The inclusion of reference strains allowed the identification of eight phena: Vibrio aestuarianus (22% of isolates), V. alginolyticus (8% of isolates), V. (Listonella) anguillarum (7% of isolates), V. salmonicida (3% of isolates), V. furnissii (1% of isolates), V. orientalis (1% of isolates), and Photobacterinm damselae subsp, piscicida (1% of isolates). Five phena were identified as V. splendidas biotype I (8% of isolates), V. mytili (7% of isolates) and Photobacrerium phosphoreum/Plesiomonas shigelloides (2% of isolates), showing a probability of identification of over 75%, according to the data matrices of Bryant et al. (1986) and Alsina and Blanch (1994a) ("Probabilislic identification of bacteria" program, version 1.10, Bryant, 1995). Some 59% of all Gram negative, facultative anaerobic, motile and oxidase positive isolates could be identified by these methods. The use of updated keys for biochemical identification of Vibrio species (Alsina & Blanch 1994b) did not provide an improved identification. The remaining 28 unidentified phena were assigned as "Vibrio spp." [FIGURE 2 OMITTED] In addition to the tests for which the same result was obtained, as described earlier for all facultative anaerobic strains, clustered facultative anaerobic isolates always displayed the same response for the following tests: growth at pH 7.5 (+), pH 9 (+), pH 10 (+), [H.sub.2]S production on TCBS (-), HDC (-), utilization of lactose in Kligler medium (-), [H.sub.2]S production in Kligler medium (-), Voges-Proskauer reaction (-). methyl red reaction (+), acid production from amygdaline, L-arabinose, ethanol, lactose, D-melibiose, L-rhamnose, salicin salicin /sal·i·cin/ (sal´i-sin) a precursor of salicylic acid, contained in the bark of the willow and poplar, that is responsible for the antiinflammatory and antipyretic effects of willow bark. or sorbitol sorbitol /sor·bi·tol/ (sor´bi-tol) a six-carbon sugar alcohol from a variety of fruits, found in lens deposits in diabetes mellitus. (-), degradation of urea or cellulose (-), and sensitivity to tetracycline (30 [micro]g/disc) (+). For each identified group (Table 2), in keeping with previous description by other authors and in addition to the responses al ready mentioned for all facultative anaerobic strains, the main characteristics were: V. aestuarianus This group was constituted by phenon 5 (94 isolates and a reference strain of V. aestuarianus). Strains included in this phenon showed a negative result for the following tests: lysine lysine (lī`sēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein. and ornithine ornithine /or·ni·thine/ (or´ni-then) an amino acid obtained from arginine by splitting of urea; it is an intermediate in urea biosynthesis. or·ni·thine n. Abbr. decarboxylation, growth at 37[degrees]C and in 10% NaCl. However, they showed a positive result for Thornley's arginine dihydrolase test, growth at 28[degrees]C, nitrate reduction, starch degradation, acid production from D-mannitol, use of citrate as sole carbon source, and susceptibility to O/129 (150 [micro]g/disc). V. splendidus biotype I This group included phenon 1 (22 isolates) and phenon 17 (12 isolates). These bacterial strains were unable to grow at 37[degrees]C, in 10% NaCl or in crystal violet crystal violet n. A dye derived from gentian violet that is used as a general biological stain, an acid-base indicator, and an agent against infection by bacteria, fungi, pinworms, and other parasites. . They were unable to use [beta]-alanine, inulin inulin /in·u·lin/ (in´ul-in) a starch occurring in the rhizome of certain plants, yielding fructose on hydrolysis, and used in tests of renal function. in·u·lin n. , L-lysine, malonate, L-phenylalanine, propanol, or L-tryptophan as sole carbon source. They showed negative responses for ornithine or lysine decarboxylase tests, and positive results for acid production from D-cellobiose, D-mannitol and ribose, use of L-proline as sole carbon source, and growth at 28[degrees]C. V. alginolyticus This group was formed by phenon 21 (33 isolates and a reference strain of V. alginolyticus). Isolates included in this phenon showed a negative result for Thornley's arginine dihydrolase test and use of propanol as sole carbon source. However, they were able to carry out nitrate reduction, hydrolysis of casein, indole production, growth on 10% NaCl and at 44[degrees]C, lysine decarboxylation, acid production from sucrose or D-trehalose, use of acetate, [beta]-alanine, DL-alanine, L-arginine or citrate as sole carbon source. They showed positive results for catalase catalase /cat·a·lase/ (kat´ah-las) a hemoprotein enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen, protecting cells. test and a variable result for ornithine decarboxylation. V. mytili This group was constituted by phenon 7 (10 isolates) and phenon 14 (22 isolates). These strains showed negative responses in tests for growth at 37[degrees]C. lysine or ornithine decarboxylation, hydrolysis of DNA or gelatin and acid production from D-mannose. They showed a positive result for Thornley's arginine dihydrolase test and acid production from sucrose. V. (Listonella) anguillarum This group included phenon 4 (12 isolates and the reference strain of V. (Listonella) anguillarum NCIMB 571) and phenon 6 (17 isolates and the reference strain of V. (Listonella) anguillarum 43-F). These strains showed negative responses in tests for ornithine and lysine decarboxylase, growth in media containing crystal violet or 10% NaCl, growth at 37[degrees]C and use of [beta]-alanine, malonate, or propanol as sole carbon sources. They showed positive results in tests for catalase, nitrate reduction, indole production, Thornley's arginine dihydrolase, hydrolysis of starch or casein, and the use of L-proline as sole carbon source. V. salmonicida Phenon 18 (13 isolates and a reference strain of V. salmonicida). These strains were unable to carry out lysine or ornithine decarboxylation, growth in 10% NaCl or at 37[degrees]C and hydrolysis of esculin or gelatin. They were able to grow at 4[degrees]C and sensitive to O/129 (150 [micro]g/disc). V. furnissii Phenon 22 (2 isolates and a reference strain of V. furnissii). These isolates had negative responses for the following tests: growth at 4[degrees]C, in 10% NaCl or in crystal violet, ornithine or lysine decarboxylation, hydrolysis of chondroitin, acid production from D-cellobiose, and the use of malonate as sole carbon source. They showed positive results for Thornley's arginine dihydrolase test, nitrate reduction, catalase test, hydrolysis of starch or casein, acid production from D-mannitol or D-trehalose and use of L-arginine, L-proline, L-serine, or succinate as sole carbon source. V. orientalis Phenon 29 (2 isolates and a reference strain of V. orientalis). These strains showed negative results for growth at 37[degrees]C and ornithine decarboxylation tests. However, they were able to grow at 28[degrees]C; in 3.5% or 5% NaCl; showed positive results on indole production and nitrate reduction tests; were able to produce acid from D-fructose and sucrose; were sensitive to O/129 (150 [micro]g/ disc); and could use pyruvate as sole carbon source. Ph. Damselae subsp. Piscicida Phenon 24 (1 isolate and a reference strain of Ph. damselae subsp. piscicida). Both strains were unable to grow at 4[degrees]C, on TCBS inedium, carry out ornithine or lysine decarboxylation, indole production, hydrolysis of gelatin, acid production from D cellobiose cellobiose a simple polysaccharide composed of two molecules of glucose and formed by the digestion of cellulose by cellulase. , glycerol, D-mannose, sucrose, or D-trehalose. Both strains showed positive results for the following tests: growth in 3.5% NaCl, catalase, and O/129 (150 [micro]g/disc) susceptibility. The following 12 tests (Table 2) are proposed as discriminatory and useful for the biochemical identification of Gram negative, motile, oxidase positive, methyl red positive, facultative anaerobic, and able to grow at 22[degrees]C strains associated with the culture of Galician bivalve mollusks: Thornley's arginine dihydrolase, lysine decarboxylase (Moeller medium), indole production, growth in different NaCl concentrations (0.5% and 7% NaCl), nitrate reduction, growth at 4[degrees]C, acid production from D-fructose, sucrose and D-trehalose, and the use of citrate and pyruvate as sole carbon source. Bacterial distribution Vibrio species were the predominant bacterial strains associated with the culture of bivalve mollusks (Table 3). Bacterial strains isolated from the seed stage in clams were identified as V. aestuarianus (facultative anaerobic isolates) or Pseudoalteromonas spp. (aerobic isolates). This may be due to the low number of bacterial strains isolated at this stage. The only aerobic strains isolated from the larval and reproductive stages in clams were identified as belonging to the genus Shewanella. The seed and reproductive stages of Ostrea edulis, the latter with the highest number of isolates, showed Vibrio, Shewanella, and Pseudoalteromonas species. A large variety of aerobic strains and different Vibrio species could be identified in the larval stage larval stage - Describes a period of monomaniacal concentration on coding apparently passed through by all fledgling hackers. Common symptoms include the perpetration of more than one 36-hour hacking run in a given week; neglect of all other activities including usual basics like of Ostrea edulis. Species of Vibrio, Photobacterium, Pseudoalteromonas, and Pseudomonas were identified from water samples. The presence of the 2 main identified species (V. aestuarianus and V. splendidus biotype I) in water samples and associated with differcut phases in the culture of oysters demonstrates the influence of the microflora microflora /mi·cro·flo·ra/ (-flor´ah) the microscopic vegetable organisms of a special region. Microflora The bacterial population in the intestine. in the surrounding water. Aerobic strains with low-NaCl requirements for growth and able to tolerate concentrations of up to 7% (w/v) NaCl were isolated from clams, oysters, water, and phytoplankton samples, revealing the influence of living in estuarine es·tu·a·rine adj. 1. Of, relating to, or found in an estuary. 2. Geology Formed or deposited in an estuary. Adj. 1. estuarine - of or relating to or found in estuaries estuarial water. Excluding samples isolated from phytoplankton, where facultative anaerobic strains were not detected, each remaining sample showed bacterial strains up to now unidentified. DISCUSSION A comparison between aerobic and facultative anaerobic strains reveals the existence of a wide diversity among the aerobic isolates. Although 352 facultative anaerobic isolates were clustered in 41 phena (see Fig. 2), and one already contained 94 of these isolates, only half of the aerobic strains could be clustered: 52 isolates in 23 phena (see Fig. 1). This shows a proportion of 8.59 facultative anaerobic strains per phenon, versus a ratio of 2.26 aerobic strains per phenon. For facultative anaerobic strains the isolates were very similar, unlike the aerobic isolates (Ortigosa et al. 1994b, Pujalte et al. 1999, Macian et al. 2000). Shewanella, Pseudoalteromonas and Pseudomonas were the main aerobic bacteria groups and Vibrio aestuarianus, V. splendidus biotype I, V. alginolyticus, V. mytili, and V. (Listonella) anguillarum were the predominant facultative anaerobic bacteria identified in this study as being associated with the culture of bivalve mollusks in Galicia. Other authors have identified these microorganisms from oysters, clams, and other bivalve mollusks with a different predominance of species: Ortigosa et al. (1994b) studied aerobic, gram negative bacteria associated with oysters and surrounding water in Valencia (Spain), identifying species of Alteromonas, Shewanella, Deleya, Flavobacterium, Oceanospirillum, Pseudomonas, and marine Agrobacterium-like organisms. Montilla et al. (1994) found that the most frequent Vibrio species associated with clams, oysters, and mussels in nurseries at the Ebro Delta (Spain) was V. fluvialis followed by V. pelagius, V. tubiashii, V. splendidus, V. alginolyticus and V. parahaemolyticus. Hariharan et al. (1995) studied bacteria associated with oysters and mussels from six river systems in Prince Edward Island Prince Edward Island, province (2001 pop. 135,294), 2,184 sq mi (5,657 sq km), E Canada, off N.B. and N.S. Geography One of the Maritime Provinces, Prince Edward Island lies in the Gulf of St. (Canada); they identified V. (Listonella) anguillurum as the most common species from oysters and V. alginolyticus, and V. splendidus as the most common from mussels, with the second largest group of bacteria being Pseudomonas/Shewanella. Pujalte et al. (1999) studied bacteria associated with the Spanish Mediterranean coast and found that V. splendidus and V. harveyi were the main bacteria associated with oysters, whilst Halomonas, Alteromonas and Pseudomonas were the principal groups identified from seawater seawater 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. . Maugeri et al. (2000) studied brackish brack·ish adj. 1. Having a somewhat salty taste, especially from containing a mixture of seawater and fresh water: "You could cut the brackish winds with a knife/Here in Nantucket" waters and mussels in the northeast of Sicily (Italy) and identified the following Vibrio species: V. fluvialis, V. alginolyticus, V. parahaemolyticus, V. vultificus, and V. mimicus. Castro et al. (2002) found that the main Vibrio species associated with cultured Manila clams from the Atlantic coast of south-western Spain were V. tubiashii, V. splendidus biotype I, and V. harveyi. Our results are in agreement with all the above mentioned studies, which show a clear prevalence of different species of genus Vibrio Noun 1. genus Vibrio - a genus of bacteria bacteria genus - a genus of bacteria family Spirillaceae, Spirillaceae - rigid spirally curved elongate bacteria vibrio, vibrion - curved rodlike motile bacterium , followed by a different dominance of aerobic strains. This demonstrates the considerable influence of the specific natural conditions of each region on the colonization of cultures of bivalve mollusks. The characteristics described in this study for each identified group ale consistent with results previously reported by other authors for facultative anaerobic (West et al. 1986, Pujalte et al. 1993, Alsina & Blanch 1994a, Cerda-Cuellar et al. 1997, Thyssen et al. 1998) and aerobic strains (Bowman 1998, Sawabe et al. 1998, Venkateswaran et al. 1999), whom groups establishment allowed a great identification but only up to a genus level. As shown in Tables 1 and 2, the results of this study make it possible to improve the characterization of Vibrio species and aerobic isolates. The set of tests selected for identifying the Vibrio species obtained from the cultures of bivalve mollusks in Galicia are similar to those previously reported by other authors (West et al. 1986; Pujalte et al. 1993, Alsina & Blanch 1994a, Cerda-Cuellar et al. 1997, Thyssen et al. 1998, Zorrilla et al. 1999). We have identified a single isolate of Ph. damselae subsp. piscicida, a causative caus·a·tive adj. 1. Functioning as an agent or cause. 2. Expressing causation. Used of a verb or verbal affix. caus agent of pasteurellosis Pasteurellosis A variety of infectious diseases caused by the coccobacilli Pasteurella multocida and P. haemolytica; the term also applies to diseases caused by any Pasteurella species. (Zorrilla et al. 1999), associated with water samples (see Table 3). Also, V. (Listonella) anguillarum, a 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. for fish, shellfish, bivalve mollusks, and other marine animals (Farmer & Hickman Brenner 1992), was isolated from oyster larvae Larvae, in Roman religion Larvae: see lemures. and reproductive oysters. Nevertheless, we could detect neither V. tublashii, a pathogen of oysters and clams (Hada et al. 1984) nor V. tapetis, the causative agent of the brown ring disease in clams (Borrego et al. 1996). Although previously associated with diseased wild octopus in NW of Spain (Farto et al. 2003), in our study, undertaken in the same area, V. lentus could not be isolated from bivalve mollusks cultures. V. alginolyticus, a potential pathogen for humans and shellfish, was isolated from clam larvae and seeds and reproductive oysters. The pathogens V. vulnificus and V. parahaemolyticus were not detected, even though they are common inhabitants
The game is based loosely on the concepts from SameGame. of estuarine and marine environments and are frequently isolated from seawater and seafood (Maugeri et al. 2000). This concurs with the results of studies undertaken on oysters from the Mediterranean Sea Mediterranean Sea [Lat.,=in the midst of lands], the world's largest inland sea, c.965,000 sq mi (2,499,350 sq km), surrounded by Europe, Asia, and Africa. Geography The Mediterranean is c.2,400 mi (3,900 km) long with a maximum width of c. (Ortigosa et al. 1994a, Pujalte et al. 1999), but contrasts with the abundance of these species in oysters from other geographical areas, such as the United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area. (O'Neill et al. 1990, Hlady et al. 1993, Wright et al. 1996) or Japan (Hor et al. 1995). The human pathogens V. fluvialis and V. metschnikovii were not detected. However, 43% of aerobic strains and 41% of facultative anaerobic strains remain unidentified. In phenon 37 (see Fig. 2), seven facultative anaerobic isolates, associated with the reproductive stage in clams, showed a different identification depending on the data matrix used: they were identified as Plesiomonas shigelloides using the matrix of Bryant et al. (1986), or as Photobacterium phosphoreum Photobacterium phosphoreum or Vibrio phosphoreum is a Gram-negative luminescent bacterium living in symbiosis with marine organisms. It can emit bluish-green light (490 nm) thanks to a chemical reaction between FMN, luciferin and molecular oxygen catalysed by an enzyme according to that of Alsina and Blanch (1994a). The probability level of being one or the other species was very high, 98%, for both species. It was not possible to differentiate between these two species with the 92 tests used in this study. More standard tests and molecular techniques should be included for a definite confirmation. This is not an ecological study about variability through time, but the high number of strains we have isolated from bivalve mollusks cultures in different geographical areas during a long time period, as well as the method we have used to obtain them, gives these isolates a representative value of the most frequent culturable bacteria associated with the culture of bivalve mollusks in the NW of Spain. Numerical taxonomy is a well accepted method to search for differential phenotipic characteristics among close taxonomic tax·o·nom·ic also tax·o·nom·i·cal adj. Of or relating to taxonomy: a taxonomic designation. tax strains. This allows a rapid presumptive identification that is very useful in diagnostics and sanitarian sanitarian /san·i·tar·i·an/ (san?i-tar´e-an) one skilled in sanitation and public health science. san·i·tar·i·an n. A public health or sanitation expert. control analyses. All discriminatory tests are presented to provide a rapid presumptive guide for identifying environmental and the most frequent pathogenic bacterial isolates from cultures of Galician bivalve mollusks. This guide has proved to be very useful for differentiating the high bacterial variability that we have found in this study.
TABLE 1.
Differential characteristics of groups identified as
containing aerobic isolates.
Group 1 Group 2
Test N = 16 N = 15
ADH 12.5 (-) 0 -
Glucose oxidation 0 - 6.7 -
KIA/[H.sub.2]S 50 v 0 -
Nitrate reduction 93.7 + 53.3 v
Growth at:
4[degrees]C 62.5 v 66.7 v
10[degrees]C 100 + 93.3 +
37[degrees]C 0 - 46.7 v
44[degrees]C 0 - 6.7 -
pH 10 100 + 86.7 (+)
Growth in:
0.5% NaCl 6.2 - 93.3 +
5% NaCl 43.7 v 100 +
7% NaCl 0 - 100 +
10% NaCl 0 - 33.3 v
Crystal violet 18.7 (-) 6.7 -
TCBS Agar 81.2 (+) 0 -
TCBS (yellow) 37.5 v 0 -
Acid from:
D-galactose 0 - 7.7 -
D-mannose 13.3 (-) 13.3 (-)
Degradation of:
Starch 43.7 v 6.7 -
Esculin 75 (+) 46.7 v
Use as sole carbon source:
acetate 18.7 (-) 93.3 +
[beta]-alanine 0 - 86.7 (+)
DL alanine 18.7 (-) 93.3 +
L-arginime 0 - 80 (+)
Glycine 6.2 - 46.7 v
Inulin 0 - 46.7 v
L-lysine 0 - 100 +
Malonate 0 - 93.3 +
L phenylalanine 0 - 46.7 v
L-proline 6.2 - 100 +
Propanol 18.7 (-) 80 (+)
Pyruvate 37.5 v 100 +
L-serine 6.2 - 73.3 (+)
Succinate 18.7 (-) 73.3 (+)
L-tartrate 0 - 46.7 v
L-tryptophan 0 - 53.3 v
Uracil 0 - 53.3 v
Sensitivity to:
O/129 (150 [micro]g) 50 v 35.7 v
Tetracycline (30 [micro]g) 85.7 (+) 100 +
Group 3 Group 4 Group 5
Test N = 15 N = 10 N = 2
ADH 0 - 40 v 0 -
Glucose oxidation 0 - 40 v 0 -
KIA/[H.sub.2]S 0 - 0 - 0 -
Nitrate reduction 13.3 (-) 70 (+) 100 +
Growth at:
4[degrees]C 26.7 (-) 30 v 0 -
10[degrees]C 66.7 v 80 (+) 100 +
37[degrees]C 20 (-) 50 v 100 +
44[degrees]C 0 - 40 v 0 -
pH 10 46.7 v 100 + 100 +
Growth in:
0.5% NaCl 13.3 (-) 80 (+) 100 +
5% NaCl 100 + 100 + 50 v
7% NaCl 93.3 + 60 v 0 -
10% NaCl 46.7 v 40 v 0 -
Crystal violet 0 - 30 (-) 50 v
TCBS Agar 0 - 20 (-) 0 -
TCBS (yellow) 0 - 0 - 0 -
Acid from:
D-galactose ND ND 33.3 v 0 -
D-mannose ND ND 33.3 v 0 -
Degradation of:
Starch 80 (+) 40 v 0 -
Esculin 41.7 v 10 - 0 -
Use as sole carbon source:
acetate 60 v 90 + 100 +
[beta]-alanine 13.3 (-) 40 v 100 +
DL alanine 60 v 30 (-) 100 +
L-arginime 60 v 50 v 50 v
Glycine 73.3 (+) 40 v 100 +
Inulin 0 - 10 - 0 -
L-lysine 26.7 (-) 30 (-) 100 +
Malonate 6.7 - 30 (-) 100 +
L phenylalanine 53.3 v 40 v 100 +
L-proline 80 (+) 90 + 100 +
Propanol 26.7 (-) 50 v 100 +
Pyruvate 66.7 v 70 (+) 100 +
L-serine 66.7 v 50 v 100 +
Succinate 46.7 v 60 v 50 v
L-tartrate 0 - 20 (-) 50 v
L-tryptophan 6.7 - 30 (-) 50 v
Uracil 13.3 (-) 10 - 0 -
Sensitivity to:
O/129 (150 [micro]g) ND ND ND ND 0 -
Tetracycline (30 [micro]g) ND ND 100 + 100 +
Group 1.-Shewanella spp.; Group 2.-Low-NaCl requirements for growth;
Group 3.-Pseudoalteromonas spp.; Group 4.-Pseudomonas spp.; Group
5.-Alcaligenes denitrificans.
Discriminatory tests for the biochemical identification of strains
isolated from bivalve molluscs that are Gram negative, motile,
oxidase positive, aerobic and able to grow al 22[degrees]C strains
are expressed in bold.
Criteria (data are expressed in percentage of positive results):
ND: No data: +: Positive result ([greater than or equal to] 90% of
positive results); -: Negative result ([less 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).
TABLE 2.
Differential characteristics of phena identified as containing
facultative anaerobic isolates.
Phenon 1 Phenon 4 Phenon 5
Test N = 22 N = 13 N = 95
Catalase 100 + 92.3 + 34.7 v
ADH 95.5 + 100 + 100 +
ODC 0 - 0 - 0 -
LDC 0 - 0 - 3.2 -
Nitrate reduction 100 + 100 + 100 +
Indole production 4.5 - 100 + 28.4 (-)
[alpha]-haemolysis 18.2 (-) 0 - 1.1 -
Growth at:
4[degrees]C 90.9 + 30.8 v 55.8 v
10[degrees]C 95.5 + 100 + 100 +
28[degrees]C 100 + 92.3 + 98.9 +
37[degrees]C 0 - 0 - 3.2 -
44[degrees]C 0 - 0 - 3.2 -
Growth in:
0.5% NaCl 0 - 0 - 6.3 -
3.5% NaCl 100 + 100 + 27.4 (-)
5% NaCl 100 + 100 + 28.4 (-)
7% NaCl 68.2 v 38.5 v 35.8 v
10% NaCl 4.5 - 0 - 0 -
Crystal violet 0 - 0 - 2.1 -
TCBS Agar 100 + 100 + 98.9 +
TCBS (yellow) 50 v 76.9 (+) 74.7 (+)
Acid from:
D-cellobiose 100 + 76.9 (+) 89.5 (+)
D-fructose 77.3 (+) 100 + 27.4 (-)
D-galactose 31.8 v 23.1 (-) 25.3 (-)
Glycerol 0 - 0 - 24.2 (-)
D-mannitol 81.8 (+) 76.9 (+) 86.3 (+)
D-mannose 54.5 v 100 + 88.4 (+)
Ribose 100 + 100 + 95.8 +
Sucrose 27.3 (-) 61.5 v 14.7 (-)
D-trehalose 50 v 92.3 + 91.6 +
Degradation of:
Starch 95.5 + 100 + 100 +
Chitin 0 - 15.4 (-) 2.1 -
Gelatin 54.5 v 76.9 (+) 23.2 (-)
Casein 100 + 100 + 96.8 +
DNA 0 - 53.9 v 8.4 -
Lecithin 86.4 (+) 84.6 (+) 62.1 v
Chondroitin 50 v 76.9 (+) 2.1 -
Esculin 50 v 15.4 (-) 77.9 (+)
Agar 0 - 11.1 (-) 2.1 -
Use as sole carbon source:
Acetate 90 + 0 - 0 -
[beta]-alanine 4.5 - 0 - 0 -
DL-alanine 95.5 + 7.7 - 30.5 v
L-arginine 40.9 v 0 - 13.7 (-)
Citrate 100 + 7.7 - 82.1 (+)
Glycine 86.4 (+) 76.9 (+) 15.8 (-)
Inulin 0 - 0 - 1.1 -
L-lysine 4.5 - 7.7 - 0 -
Malonate 4.5 - 0 - 0 -
L-phenylalanine 0 - 0 - 0 -
L-proline 95.5 + 76.9 (+) 96.8 +
Propanol 18.2 (-) 0 - 1.1 -
Pyruvate 86.4 (+) 69.2 v 87.4 (+)
L-serine 90.9 + 61.5 v 65.3 v
Succinate 95.5 + 7.7 - 74.7 (+)
L-tartrate 4.5 - 0 - 0 -
L-tryptophan 0 - 0 - 1.1 -
Uracil 0 - 7.7 - 0 -
Sensitivity to:
O/129 (150 [micro]g) 61.9 v 46.2 v 81.1 (+)
Catalase 54.5 v 100 + 100 +
ADH 16.7 (-) 100 + 14.7 (-)
ODC 0 - 0 - 47.1 v
LDC 0 - 0 - 79.4 (+)
Nitrate reduction 100 + 100 + 100 +
Indole production 100 + 92.9 + 97.1 +
[alpha]-haemolysis 0 - 0 - 2.9 -
Growth at:
4[degrees]C 0 - 100 + 17.7 (-)
10[degrees]C ND ND 100 + 10 -
28[degrees]C 100 + 100 + 10 -
37[degrees]C 0 - 7.1 - 100 +
44[degrees]C 0 - 0 - 91.2 +
Growth in:
0.5% NaCl 0 - 100 + 100 +
3.5% NaCl 100 + 100 + 100 +
5% NaCl 100 + 100 + 100 +
7% NaCl 58.3 v 78.6 (+) 100 +
10% NaCl 0 - 14.3 (-) 76.5 (+)
Crystal violet 8.3 - 7.1 - 82.4 (+)
TCBS Agar 100 + 100 + 97.1 +
TCBS (yellow) 100 + 92.9 + 88.2 (+)
Acid from:
D-cellobiose 100 + 0 - 0 -
D-fructose ND ND 42.9 v 94.1 +
D-galactose 50 v 0 - 0 -
Glycerol 25 (-) 42.9 v 23.5 (-)
D-mannitol 91.7 + 0 - 32.4 v
D-mannose 100 + 28.6 v 35.3 v
Ribose 100 + 35.7 v 61.8 v
Sucrose 100 + 92.9 + 79.4 (+)
D-trehalose ND ND 92.9 + 100 +
Degradation of:
Starch 0 - ND ND ND ND
Chitin 0 - 7.1 - 0 -
Gelatin 8.3 - 0 - 2.9 -
Casein 0 - 100 + 79.4 (+)
DNA 0 - 0 - 35.3 v
Lecithin 0 - ND ND ND ND
Chondroitin 50 v 0 - 0 -
Esculin 100 + 0 - 44.1 v
Agar 0 - 0 - 0 -
Use as sole carbon source:
Acetate 0 - 28.6 (-) 97.1 +
[beta]-alanine 0 - 42.9 v 73.5 (+
DL-alanine ND ND 85.7 (+) 100 +
L-arginine ND ND 14.3 (-) 88.2 (+)
Citrate 0 - 85.7 (+) 73.5 (+)
Glycine 66.7 v 64.3 v 29.4 (-)
Inulin 0 - 71.4 (+) 8.8 -
L-lysine 0 - 42.9 v 55.9 v
Malonate 0 - 64.3 v 76.5 (+)
L-phenylalanine 0 - 14.3 (-) 82.4 (+)
L-proline 83.3 (+) 100 + 29.4 (-)
Propanol 0 - 71.4 (+) 26.5 (-)
Pyruvate 58.3 v 100 + 29.4 (-)
L-serine 0 - 100 + 29.4 (-)
Succinate 0 - 100 + 29.4 (-)
L-tartrate 0 - 28.6 (-) 52.9 v
L-tryptophan 0 - 64.3 v 67.7 v
Uracil 41.7 v 50 v 67.7 v
Sensitivity to:
O/129 (150 [micro]g) 75 (+) 85.7 (+) 82.4 (+)
Phenon 6 Phenon 7 Phenon 14
Test N = 18 N = 10 N = 22
Catalase 94.4 + 90 + 13.6 (-)
ADH 100 + 70 (+) 100 +
ODC 0 - 0 - 0 -
LDC 0 - 0 - 0 -
Nitrate reduction 100 + 80 (+) 13.6 (-)
Indole production 100 + 50 v 0 -
[alpha]-haemolysis 0 - 0 - 45.5 v
Growth at:
4[degrees]C 94.4 + 70 (+) 0 -
10[degrees]C 100 + 100 + 95 +
28[degrees]C 100 + 80 (+) 10 -
37[degrees]C 0 - 0 - 5 -
44[degrees]C 0 - 0 - 0 -
Growth in:
0.5% NaCl 88.9 (+) 100 + 100 +
3.5% NaCl 100 + 100 + 100 +
5% NaCl 94.4 + 100 + 100 +
7% NaCl 38.9 v 0 - 0 -
10% NaCl 5.6 - 0 - 0 -
Crystal violet 16.7 (-) 10 - 68.2 v
TCBS Agar 100 + 100 + 100 +
TCBS (yellow) 88.9 (+) 100 + 100 +
Acid from:
D-cellobiose 0 - 0 - 0 -
D-fructose 83.3 (+) 90 + 100 +
D-galactose 5.6 - 0 - 0 -
Glycerol 0 - 10 - 0 -
D-mannitol 0 - 0 - 0 -
D-mannose 11.1 (-) 10 - 9.1 -
Ribose 16.7 (-) 0 - 31.8 v
Sucrose 72.2 (+) 70 (+) 90.9 +
D-trehalose 66.7 v 100 + 86.4 (+)
Degradation of:
Starch 72.2 (+) 100 + 100 +
Chitin 0 - 0 - 0 -
Gelatin 0 - 0 - 0 -
Casein 94.4 + 100 + 100 +
DNA 11.1 (-) 0 - 22.7 (-)
Lecithin 50 v 60 v 23.8 (-)
Chondroitin 5.6 - 0 - 0 -
Esculin 72.2 (+) 60 v 66.7 v
Agar 0 - 0 - 0 -
Use as sole carbon source:
Acetate 0 - 0 - 4.6 -
[beta]-alanine 0 - 0 - 4.6 -
DL-alanine 33.3 v 0 - 36.4 v
L-arginine 11.1 (-) 0 - 0 -
Citrate 22.2 (-) 0 - 45.5 v
Glycine 22.2 (-) 0 - 95.5 +
Inulin 0 - 0 - 0 -
L-lysine 0 - 0 - 0 -
Malonate 0 - 0 - 0 -
L-phenylalanine 0 - 0 - 0 -
L-proline 83.3 (+) 60 v 100 +
Propanol 0 - 0 - 0 -
Pyruvate 100 + 10 - 100 +
L-serine 83.3 (+) 10 - 45.5 v
Succinate 88.9 (+) 20 (-) 100 +
L-tartrate 0 - 0 - 0 -
L-tryptophan 5.6 - 0 - 0 -
Uracil 0 - 0 - 0 -
Sensitivity to:
O/129 (150 [micro]g) 94.4 + 100 + 100 +
Catalase 100 + 100 + 66.7 v
ADH 100 + 50 v 100 +
ODC 0 - 0 - 0 -
LDC 0 - 0 - 0 -
Nitrate reduction 100 + 50 v 100 +
Indole production 100 + 0 - 100 +
[alpha]-haemolysis 0 - 0 - 33.3 v
Growth at:
4[degrees]C 0 - 0 - 0 -
10[degrees]C 100 + 50 v 66.7 v
28[degrees]C 100 + 100 + 100 +
37[degrees]C 33.3 v 50 v 0 -
44[degrees]C 33.3 v 50 v 0 -
Growth in:
0.5% NaCl 100 + 50 v 100 +
3.5% NaCl 100 + 100 + 100 +
5% NaCl 100 + 100 + 100 +
7% NaCl 66.7 v 100 + 66.7 v
10% NaCl 0 - 100 + 66.7 v
Crystal violet 0 - 0 - 0 -
TCBS Agar 100 + 0 - 100 +
TCBS (yellow) 0 - 0 - 100 +
Acid from:
D-cellobiose 0 - 0 - 0 -
D-fructose 100 + 0 - 100 +
D-galactose 0 - 0 - 0 -
Glycerol 0 - 0 - 0 -
D-mannitol 100 + 0 - 0 -
D-mannose 66.7 v 0 - 33.3 v
Ribose 100 + 0 - 0 -
Sucrose 0 - 0 - 100 +
D-trehalose 100 + 0 - 66.7 v
Degradation of:
Starch 100 + 0 - 100 +
Chitin 66.7 v 0 - 33.3 v
Gelatin 33.3 v 0 - 0 -
Casein 100 + ND ND 0 -
DNA 0 - ND ND 0 -
Lecithin 66.7 v ND ND 33.3 v
Chondroitin 0 - ND ND 0 -
Esculin 0 - ND ND 33.3 v
Agar 0 - 0 - 33.3 v
Use as sole carbon source:
Acetate 33.3 v 100 + 0 -
[beta]-alanine 0 - 50 v 0 -
DL-alanine 66.7 v 100 + 0 -
L-arginine 100 + 100 + 0 -
Citrate 0 - 100 + 0 -
Glycine 100 + 50 v 0 -
Inulin 33.3 v 100 + 0 -
L-lysine 0 - 100 + 0 -
Malonate 0 - 50 v 0 -
L-phenylalanine 0 - 100 + 0 -
L-proline 100 + 100 + 0 -
Propanol 0 - 100 + 0 -
Pyruvate 100 + 100 + 100 +
L-serine 100 + 100 + 0 -
Succinate 100 + 100 + 0 -
L-tartrate 0 - 100 + 0 -
L-tryptophan 0 - 100 + 0 -
Uracil 0 - 50 v 0 -
Sensitivity to:
O/129 (150 [micro]g) ND ND 100 + 100 +
"N" indicates the number of isolates included in each phenon;
1.-Vibrio splendidus biotype I; 4.-V. (Listonella) anguillarum;
5.-V. aestuarianus; 6.-V. anguillarum: 7.-V. mytili; 14.-V. mytili:
17.-V. splendidus biotype I; 18.-V. salmonicida; 21.-V. alginolyticus;
22.-V. furnissii; 24.-Photobacterum damselae subsp. piscicida; 29.-V.
orientalis.
Discriminatory tests for the biochemical identification of strains
isolated from bivalve molluscs that are Gram negative, motile, oxidase
positive, methyl red positive, facultative anaerobic and able to grow
at 22[degrees]C are expressed in bold.
Criteria (data are expressed in percentage of positive results):
ND, No data; +, Positive result ([greater than or equal to] 90%, of
positive results); -, Negative result ([less than or equal to] 10%
of positive results); (+), Mainly positive results ([greater than or
equal to] 70% <90% of positive results); (-), Mainly negative results
([less than or equal to] 10% <30% of positive results); v, Variable
results (>30% <70% of positive results).
TABLE 3.
Origin of identified and unidentified isolates.
Clam Oyster
Identification Seed Larva Rep. * Seed
([dagger]) Vibrio aestuarianus + - - +
([dagger]) Vibrio splendidus
biotype I - - + -
([dagger]) Vibrio alginolyticus - + - +
([dagger]) Vibrio mytili - + - +
([dagger]) V. (Listonella)
anguillarum - - - -
([dagger]) Vibrio salmonicida - + - +
([dagger]) Photobacterium phos-
phoreum/Plesiomonas shigelloides - - + -
([dagger]) Vibrio furnissii - - - -
([dagger]) Vibrio orientalis - + - -
([dagger]) Ph. damselae subsp.
Piscicida - - - -
([dagger]) No. of unidentified
isolates 3 13 23 6
([double dagger]) Shewanella spp. - + + +
([double dagger]) Pseudo-
alteromonas spp. + - - +
([double dagger]) Pseudomonas spp. - - - -
([double dagger]) Alcaligenes
denitrificans - - - -
([double dagger]) Low-NaCl
requirements + - - -
([double dagger]) No. of
unidentified isolates 3 1 1 3
Total number of isolates 10 33 48 31
Clam Oyster
Identification Larva Rep. * Water Phyto *
([dagger]) Vibrio aestuarianus + + + -
([dagger]) Vibrio splendidus
biotype I + + + -
([dagger]) Vibrio alginolyticus - + - -
([dagger]) Vibrio mytili - + - -
([dagger]) V. (Listonella)
anguillarum + + - -
([dagger]) Vibrio salmonicida - + - -
([dagger]) Photobacterium phos-
phoreum/Plesiomonas shigelloides - - - -
([dagger]) Vibrio furnissii - + - -
([dagger]) Vibrio orientalis - - - -
([dagger]) Ph. damselae subsp.
Piscicida - - - -
([dagger]) No. of unidentified
isolates 19 75 11 -
([double dagger]) Shewanella spp. + + - -
([double dagger]) Pseudo-
alteromonas spp. + + + +
([double dagger]) Pseudomonas spp. + - + +
([double dagger]) Alcaligenes
denitrificans + - - -
([double dagger]) Low-NaCl
requirements + + + +
([double dagger]) No. of
unidentified isolates 12 3 4 2
Total number of isolates 80 238 43 5
([dagger]), Facultative anaerobic strains. ([double dagger]),
Aerobic strains. The "+" symbol denotes the presence of one
species in a specific sample and the "-" symbol indicates its
absence.
*, Rep. = Reproductive; Phyto., Phyloplankton.
ACKNOWLEDGMENTS This work was supported by grants PGIDIT00MAR2002PR and 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. According to the Galician Statute of Autonomy, it consists of the president, the vice-president (if necessary), and the specialized ministers (Conselleiros). . The authors thank J. Montes and CECT for the kind provision of bacterial isolates and reference strains respectively, and also E. Longo for providing the multipoint inoculator. LITERATURE CITED Alsina, M. & A. R. Blanch. 1994a. A set of keys for biochemical identification of environmental Vibrio species. J. Appl. Bacteriol. 76:79-85. Alsina, M. & A. R. Blanch. 1994b. Improvement and update of a set of keys for biochemical identification of Vibrio species. J. Appl. Bacteriol. 77:719-721. Borrego, J. J., D. Castro. A. Luque, C. Paillard, P. Maes. M. T. Garcia & A. 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Isolation and characterisation of the causative agent of pasteurellosis, Photobacterium damsela subsp, piscicida, from sole. Solea senegalensis (Kaup). J. Fish Dis. 22:167-172. J. A. GUISANDE, M. MONTES, R. FARTO, S. P. ARMADA, M. J. PEREZ AND T. P. NIETO * Area de Microbiologia, Departamento de Biologia Funcional y Ciencias de la Salud, Facultad de Ciencias, Universidad de Vigo, Vigo, Spain * Corresponding author. E-mail: mtperez@uvigo.es |
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