Responses of Pyrodinium bahamense var. compressum and associated cultivable bacteria to antibiotic treatment

Pyrodinium bahamense var. compressum is a toxic dinoflagellate that produces paralytic shellfish poisoning toxins. It is responsible for the chronic toxicity of shellfish in many coastal areas of the Philippines and other South East Asian countries. For the purpose of using antibiotic treatment to possibly generate axenic cultures and understand their growth requirements, the antibiotic tolerances of two local P. bahamense var. compressum isolates and their associated bacteria were determined. The antibacterial compounds ampicillin, chloramphenicol, ciprofloxacin, kanamycin, neomycin, penicillin G, and streptomycin were tested, as well as two antifungals, amphotericin B, and nystatin. All except chloramphenicol, amphotericin B, and nystatin were generally well-tolerated. An antibiotic mixture composed of ciprofloxacin, kanamycin, neomycin, and streptomycin completely inhibited the cultivable bacteria associated with P. bahamense var. compressum MZRVA, although epifluorescence microscopy revealed that residual bacteria were still present. From long-term tests with this antibiotic mix, it was observed that survival of isolate MZRVA post-antibiotic treatment appeared to be associated with re-growth of heterotrophic bacteria and that excess vitamins could potentially enhance dinoflagellate survival. These results suggest that the associated heterotrophic bacterial populations help support the growth of P. bahamense var. compressum MZRVA in culture and possibly in nature. This is the first report on the growth responses of P. bahamense var. compressum and associated cultivable bacteria to a variety of single and combinations of antibiotics.     

Morphology of Pyrodinium bahamense Plate (Dinoflagellata) near Isla San José, Gulf of California, Mexico

The morphology of Pyrodinium bahamense south of Isla San José in the Gulf of California, Mexico was described using light and scanning electron microscopy. Morphological and dimensional examination of whole cells and thecal patterns were conducted to discriminate between P. bahamense var. compressum and P. bahamense var. bahamense. Microscopic examinations confirmed that specimens of P. bahamense from Isla San José are closely related to P. bahamense var. bahamense. The main average dimensions were: cells 41.9 μm long, 43.8 μm wide, 55.6 μm amplitude, apical horn 7.2 μm long, and the left antapical spine 19.5 μm long. The highest cell concentration was 240 cells l⁻¹ and occurred with a bloom of Pseudo-nitzschia spp. The presence of this dinoflagellate off the east coast of the Baja California Peninsula had not been previously reported. Recently, P. bahamense from Florida tested positive for saxitoxins. Paralytic shellfish poisoning from this species has occurred in Mexican waters. Here, significant aspects of taxonomy, toxicology, life cycle, and ecology are discussed.     

Horizontal dinoflage11ate cyst distribution, sediment characteristics and benthic flux in Manila Bay, Philippines

The lateral variation of sediment properties and associated cyst content of sediment in Manila Bay were determined and their possible role/s in the occurrences of Pyrodinium bahamense Plate var. compressum (Bohm) Steidinger, Tester et Taylor toxic blooms were assessed. Manila Bay's surface sediment was determined to be silt dominated. Clay generally increased towards the coast, probably as a result of flocculation and rapid deposition upon entry of sediments from the rivers. High sand content characterized the southeastern part of the bay attributed to the greater sand inputs and relatively strong currents in this area. Bulk densities were lower in the eastern side of the bay from dilution by high organic load from sewage and urban areas. Benthic flux calculations, particularly NH3, suggest more than 50% nutrient contribution comes from sediments. In general, dinoflagellate cyst density increased from the center of the bay towards the coast, except in Pampanga Bay where it deceased near the coasts. A maximum of 23 dinoflagellate species were identified: 5 were autotrophic (Lingulodinium polyedrum (Stein) Dodge, Gonyaulax spp., Pyrophacus steinii (Schiller) Wall et Dale, Protoceratium reticulatum (Claparede et Lachmann) Butschli, and Pyrodinium bahamense var. compressum), and the rest were predominantly composed of Protoperidinium spp. and Diplopsalis spp. Heterotrophs comprised about 70% of the total cyst counts. Pyrodinium counts increased towards the northwestern part of the bay where it was the dominant autotroph species. Negative correlations were observed for live Pyrodinium cyst density and N flux, P flux, ratio of N to P and total organic carbon (TOC) content. However, areas with high N:P ratio contain abundant Pyrodinium live cysts.     

Monoxenic culture of the anaerobic ciliate Trimyema compressum Lackey

Sapropelic ciliates from anoxic mud samples were enriched and cultivated in monoculture together with natural food bacteria growing on cellulose. The ciliates lacked cytochrome oxidase and contained bluish fluorescent endosymbionts. One of the anaerobic ciliates, Trimyema compressum, contained methanogenic bacteria as was shown by methane formation. During continued cultivation, T. compressum gradually lost its endosymbionts. With SEM microscopy no episymbiotic bacteria could be detected.From enrichment cultures of T. compressum, anaerobic bacteria were isolated in pure culture. One of the strains, a Bacteroides spec., proved capable of serving as food bacteria, thus allowing establishment of monoxenic T. compressum cultures. These cultures exhibited a requirement for sterols as growth factors. The doubling time of this ciliate was 13 h at 28°C. The highest yield obtained was 2100 cells/ml.Dedicated to Holger W. Jannasch on the occasion of his 60th birthday     

Structures of Two Paralytic Shellfish Toxins,Gonyautoxins V and VI,Isolated from a Tropical Dinoflagellate,Pyrodinium bahamense var. compressa

Two paralytic shellfish toxins, gonyautoxin V and gonyautoxin VI, isolated from a tropical dinoflagellate, Pyrodinium bahamense var. compressa, were identified respectively to be derivatives of saxitoxin and neosaxitoxin with a sulfonatocarbamoyl moiety.     

Characterization and identification of bacteria isolated from micropropagated mint plants

Summary Bacterial isolates from contaminated mint shoot cultures were characterized and identified as a preliminary step in determining an elimination treatment. The 22 bacteria were characterized using biochemical and morphological tests and subjected to sensitivity tests with four antibiotics. The isolates were compared with known organisms and assigned to genera according to similarities in characteristics. Seven isolates were analyzed by fatty acid analysis carried out by a commercial laboratory. Six were classified asAgrobacterium radiobacter; eight asXanthomonas; one each asPseudomonas fluorescens, Micrococcus spp.,Corynebacterium spp., andCurtobacterium spp.; four could not be assigned to genera. Inhibition of growth of the bacteria by most antibiotics was best at pH 7.5. Minimal inhibitory concentration and minimal bactericidal concentrations of gentamicin, rifampicin, streptomycin sulfate, and Timentin varied with genotype.     

Rickettsial Agents from Parasitic Dermanyssoidea (Acari: Mesostigmata)

Mites are often overlooked as vectors of pathogens, but have been shown to harbor and transmit rickettsial agents such as Rickettsia akari and Orientia tsutsugamushi. We screened DNA extracts from 27 mites representing 25 species of dermanyssoids for rickettsial agents such as Anaplasma, Bartonella, Rickettsia, and Wolbachia by PCR amplification and sequencing. DNA from Anaplasma spp., a novel Bartonella sp., Spiroplasma sp., Wolbachia sp., and an unclassified Rickettsiales were detected in mites. These could represent mite-borne bacterial agents, bacterial DNA from blood meals, or novel endosymbionts of mites.     

Fermentation products of the anaerobic ciliate Trimyema compressum in monoxenic cultures

A new monoxenic culture of the anaerobic ciliate Trimyema compressum was established on the purple nonsulfur bacterium Rubrivivax gelatinosus. Compared with the first monoxenic culture of T. compressum on a Bacteroides species no significant difference in growth rate was observed. Using both monoxenic cultures the fermentation products of the ciliate, acetate, lactate, formate and succinate were quantified. Ammonium was another product of the ciliates metabolism. The amount of the endproducts was dependent on the amount of bacteria consumed. The ratio of carbon consumed to ammonium formed was 8.7:1 indicating incomplete degradation of nitrogenous compounds of the bacterial biomass. Under microoxic conditions no effect of oxygen on the yield of the ciliate was observed but the amount of acetate and lactate was 25% lower. Cells of T. compressum with Methanobacterium formicicum as deliberately introduced endosymbiont did not form significantly different fermentation products, however, instead of formate methane was detected as product. The yield of the ciliate was not affected. It is concluded that methanogenic bacteria deliberately introduced into the cytoplasm of T. compressum as endosymbionts are pure commensalists.     

Numerical taxonomy of fluorescent Pseudomonas associated with tomato roots

The phenetic taxonomy of 110 fluorescent bacterial strains, isolated from the roots of tomatoes and other plants was numerically studied through 97 features including 69 assimilation tests. Thirty-two reference strains of various Pseudomonas spp. were additionally included. The strains clustered into 16 clusters at the 74% similarity level when using Jaccard similarity coefficients. Almost all field strains belonged to the P. fluorescens/P. putida-complex while none clustered with P. syringae and allied bacteria. The biovar II branch, as well as the newly described biovar VI of P. fluorescens, made up 55% and 20% respectively, of the field strains; two % were allocated to P. fluorescens biovar I and three % to biovar IV. Eleven % of the root associated strains were designated P. putida; six strains were biovar A, three strains biovar B while four strains could not be referred to any known biovar. The continuum within the P. fluorescens/P. putida-complex as well as the taxonomic status of the six biovars of P. fluorescens and the three biovars of P. putida are discussed.     

Biotransformation of isoeugenol to vanillin by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IE27 cells

The ability to produce vanillin and/or vanillic acid from isoeugenol was screened using resting cells of various bacteria. The vanillin- and/or vanillic-acid-producing activities were observed in strains belonging to the genera Achromobacter, Aeromonas, Agrobacerium, Alcaligenes, Arthrobacter, Bacillus, Micrococcus, Pseudomonas, Rhodobacter, and Rhodococcus. Strain IE27, a soil isolate showing the highest vanillin-producing activity, was identified as Pseudomonas putida. We optimized the culture and reaction conditions for vanillin production from isoeugenol using P. putida IE27 cells. The vanillin-producing activity was induced by adding isoeugenol to the culture medium but not vanillin or eugenol. Under the optimized reaction conditions, P. putida IE27 cells produced 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20 °C after a 24-h incubation in the presence of 10% (v/v) dimethyl sulfoxide.     

DNA relatedness among saturn-spored yeasts assigned to the generaWilliopsis andPichia

Saturn-spored species assigned to the generaWilliopsis andPichia were compared from extent of nuclear DNA complementarity. Of thePichia spp., four were recognized as distinct taxa:P. dispora, P. saitoi, P. zaruensis andPichia sp. nov. AmongWilliopsis spp., the following were accepted:W. californica, W. mucosa comb. nov.,W. pratensis, W. saturnus var.saturnus, W. saturnus var.mrakii comb. nov.,W. saturnus var.sargentensis comb. nov.,W. saturnus var.subsufficiens comb. nov. andWilliopsis sp. nov. The newPichia andWilliopsis species are described elsewhere. Moderate (36–68%) DNA relatedness was detected between the formerPichia sargentensis and varieties ofW. saturnus again demonstrating that nitrate assimilation is not a reliable criterion for separating yeast species.     

Morphological differences and molecular phylogeny of freshwater blooming species, Peridiniopsis spp. (Dinophyceae) from China

In 2008–2010, several freshwater dinoflagellate blooms caused by Peridiniopsis spp. were observed in China. P. penardii and P. niei sampled from various geographical localities were examined by means of light and scanning electron microscopy. After comparing morphological and molecular differences, the new freshwater variety Peridiniopsis penardii var. robusta var. nov. (Peridiniales, Dinophyceae) found in Manwan Reservoir, Yunnan Province was described. The new variety differed from P. penardii since it possessed numerous robust antapical spines and a conspicuous apical spine. Molecular phylogenetic analyses based on SSU, LSU and ITS indicated P. niei, P. penardii and P. penardii var. robusta were closely related with P. kevei, and clustered into a monophyletic clade. The new variety possessed an endosymbiotic diatom which was similar to P. penardii and P. kevei, whereas the endosymbiont was not present in cells of P. niei. The endosymbiont SSU and ITS phylogenies showed that the endosymbionts of these three dinoflagellates were closely related to members of Thalassiosirales. Furthermore it was concluded that the endosymbionts might originate from Discostella-like species.     

Novel actinobacteria from marine sponges

Actinobacteria exclusively within the sub-class Acidimicrobidae were shown by 16S rDNA community analysis to be major components of the bacterial community associated with two sponge species in the genus Xestospongia. Four groups of Actinobacteria were identified in Xestospongia spp., with three of these four groups being found in both Xestospongia muta from Key Largo, Florida and Xestospongia testudinaria from Manado, Indonesia. This suggests that these groups are true symbionts in these sponges and may play a common role in both the Pacific and Atlantic sponge species. The fourth group was found only in X. testudinaria and was a novel assemblage distantly related to any previously sequenced actinobacterial clones. The only actinobacteria that were obtained in initial culturing attempts were Gordonia, Micrococcus and Brachybacterium spp., none of which were represented in the clone libraries. The closest cultured actinobacteria to all the Acidimicrobidae clones from Xestospongia spp. are Microthrix parvicella and Acidimicrobium spp. Xestospongia spp. can now be targeted as source material from which to culture novel Acidimicrobidae to investigate their potential as producers of bioactive compounds. Isolation of sponge-associated Acidimicrobidae will also make it possible to elucidate their role as sponge symbionts.     

Cell hydrophobicity of <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. and <Emphasis Type="Italic">Bacillus</Emphasis> spp. bacteria and hydrocarbon biodegradation in the presence of Quillaya saponin

Biodegradation and hydrophobicity of Pseudomonas spp. and Bacillus spp. strains were tested at different concentrations of the biosurfactant Quillaya saponin. A model mixture of hydrocarbon (dodecane and hexadecane) was used for estimating the influence of surfactants on biodegradation. The bacterial adhesion to hydrocarbon method for determination of bacterial cell surface hydrophobicity was exploited. Among the tested bacterial strains the higher hydrophobicity was noticed for Pseudomonas aeruginosa TK. The hydrophobicity of this strain was 84%. The highest hydrocarbon biodegradation was observed for P. aeruginosa TK (49%) and Bacillus subtilis (35%) strains after 7 days of experiments. Generally the addition of Quillaya saponin increased hydrocarbon biodegradation remarkably. The optimal concentration proved to be 80 mg l⁻¹. The degree of hydrocarbon biodegradation was 75% for P. aeruginosa TK after the addition of saponin. However the most significant increase in biodegradation after addition of Quillaya saponin was in the case of P. aeruginosa 25 and Pseudomonas putida (the increase of biodegradation from 21 to 52% and from 31 to 66%, respectively). It is worth mentioning that decrease of hydrophobicity is correlated with the best biodegradation by P. aeruginosa strain. For the remaining strains, no significant hydrophobicity changes in relation to the system without surfactant were noticed.     

A fish kill of massive proportion in Kuwait Bay, Arabian Gulf, 2001: the roles of bacterial disease, harmful algae, and eutrophication

In August and September 2001, Kuwait Bay, a semi-enclosed embayment of the Arabian Gulf, experienced a massive fish kill involving over >2500 metric tons of wild mullet (Liza klunzingeri), due to the bacterium Streptococcus agalactiae. In the Bay, this event was preceded by a small fish kill (100–1000 dead fish per day) of gilthead sea bream (Sparus auratus) in aquaculture net pens associated with a bloom of the dinoflagellate Ceratium furca. Sea bream were found to be culture positive for S. agalactiae, but did not show any visible signs of disease. Unusually warm temperatures (up to 35 °C) and calm conditions prevailed during this period. As the wild fish kill progressed, various harmful algae were observed, including Gymnodinium catenatum, Gyrodinium impudicum, and Pyrodinium bahamense var. compressum. Cell numbers of G. catenatum and G. impudicum exceeded 10⁶ l⁻¹ in some locations. All fish tested below the limits of detection for paralytic shellfish poisoning (PSP) and brevetoxins. Clams (Circe callipyga) were positive for PSP but at levels below regulatory limits. Nutrient concentrations, both inorganic and organic, were highly variable with time and from site to site, reflecting inputs from sewage outfalls, the aquaculture operations, a high biomass of decomposing fish, and other sources. It is hypothesized that many factors contributed to the initial outbreak of the bacterial disease, including unusual warm and calm conditions. The same factors, as well as enriched nutrient conditions, also apparently were conducive to the subsequent HAB outbreaks. The detection of PSP, while below regulatory limits, warrants further monitoring to protect human health.     

Performance evaluation of potent phosphate solubilizing bacteria in potato rhizosphere

Three phosphate solubilizing bacterial isolates identified as Pantoea agglomerans strain P5, Microbacterium laevaniformans strain P7 and Pseudomonas putida strain P13 were assessed for mutual relationships among them, competitiveness with soil microorganisms and associations with plant root using luxAB reporter genes for follow-up studies. Synergism between either P. agglomerans or M. laevaniformans, as acid-producing bacteria, and P. putida, as a strong phosphatase producer, was consistently observed both in liquid culture medium and in root rhizosphere. All laboratory, greenhouse and field experiments proved that these three isolates compete well with naturally occurring soil microorganisms. Consistently, the combinations of either P. agglomerans or M. laevaniformans strains with Pseudomonas putida led to higher biomass and potato tuber in greenhouse and in field trials. It is conceivable that combinations of an acid- and a phosphatase-producing bacterium would allow simultaneous utilization of both inorganic and organic phosphorus compounds preserving the soil structure.     

Regulation of amino acid and glucose dissimilation in so-called ammonifiers and in other soil microorganisms

Pseudomonas acidovorans and P. putida, isolated from an enrichment culture with casein hydrolysate, and Agrobacterium radiobacter and Torulopsis sp., isolated from a glucose enrichment, were compared with respect to the physiology of ammonification. Decreasing ammonifying ability as well as increasing repression of the synthesis of amino acid degrading enzymes by glucose were found in the above order of organisms. In degradation sequences, observed with P. putida and A. radiobacter as test organisms, substances dissimilated prior to others had both, enhancing and repressing effects on the oxidation of the other compounds. This fact was parallelled by the observation, that in these two bacteria, glucose and single amino acids, when added to the same medium, exerted mutual repression of the synthesis of catabolic enzymes of their partners. The ecological significance of this type of regulation has been discussed.     

Isolation and identification ofMicrococcus roseus andPlanococcus sp. from schirmacher oasis,antarctica

Five cultures isolated from soil samples collected in Schirmacher oasis, Antarctica, have been identified as members of the familyMicrococcaceae, with 3 belonging to the genusMicrococcus and two toPlanococcus. The 3Micrococcus isolates (37R, 45R and 49R) were red-pigmented and h a d ∼ 75 mol% G + C in their DNA; they were identified asMicrococcus roseus. The twoPlanococcus isolates (30Y and Lz3OR) were yellow and orange in colour, and had 43.5 and 40.9 mol % G + C in their DNA respectively; they were identified asPlanococcus sp.     

The first study on the bacterial flora of the european spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae)

The European spruce bark beetle, Dendroctonus micans Kugelann (Coleoptera, Scolytidae), is one of the most serious pests of oriental spruce (Picea orientalis L.) in Turkey. In this study, we investigated bacterial flora of D. micans collected from different populations of the forests of Eastern Black Sea Region of Turkey from 2002 to 2004. Seven different bacteria were isolated from healthy, diseased and dead specimens based on the color of colony and morphology. According to morphological, physiological and biochemical properties, metobolic enyzme profile by BIOLOG microtiter plate system, and total cellular fatty acid profile by Microbial Identification System (MIS), isolates were identified as Micrococcus luteus, Bacillus thuringiensis subsp. morrisoni, Serratia grimesii, Enterobacter cloaceae, Enterobacter intermedius, Streptococcus sp. and Pseudomonas putida. This is the first study on the bacterial flora of D. micans.     

Biological control of bacterial spot of tomato under field conditions at several locations in North America

Following the relatively successful biological control of bacterial speck of tomato under field conditions at several locations (Phytopathology 92 (2002) 1284), similar selection and testing strategies were employed in an effort to isolate an effective biological control agent for bacterial spot of tomato. Fifty potential biological control agents were isolated from tomato foliage in Alabama (AL) and Florida (FL) and tested under greenhouse conditions in AL for the ability to reduce the foliar severity of bacterial spot of tomato (Lycopersicon esculentum), which is caused by either Xanthomonas campestris pv. vesicatoria or Xanthomonas vesicatoria. Three pseudomonads that provided protection against bacterial speck also were included in the tests. The strains which were most efficacious (i.e., high mean percentage reduction) and consistent (i.e., low standard deviation) in reducing bacterial spot severity in repeated greenhouse experiments were selected for field experiments conducted over the period 1996–1998. Among these strains were Cellulomonas turbata BT1, which provided the highest mean reduction in disease severity [45.2% (SD = 21.0)], and Pseudomonas syringae Cit7 [36.4% (SD = 12.2)], which was the most consistent. Field experiments were conducted in Shorter, AL; Bradenton and Sanford, FL; Clinton, North Carolina; Wooster, Ohio; and London, Ontario, Canada. The highest mean reductions in severity of bacterial spot on foliage, averaged across all locations, were provided by P. syringae Cit7 [28.9% (SD = 11.6)] and Pseudomonas putida B56 [23.1% (SD = 7.4)]. The efficacy and consistency of P. syringae Cit7 against bacterial spot were very similar to those achieved against bacterial speck [28.3% (SD = 12.7)] (Phytopathology 92 (2002) 1284). Unfortunately, neither the bacterial strains nor the standard copper bactericides consistently reduced disease incidence on fruit.