Phosphatase activity and its rôle in the mineralization of organic phosphorus in coastal sea water

Alkaline phosphatase activity in sea-water samples taken from Tokyo, Sagami, and Suruga Bays in Japan was measured by a sensitive fluorometric method. There is a relationship between the phosphatase activity and bacterial biomasses in these three bays. The phosphatase-producing bacteria accounted for 40, 46, and 41% of heterotrophic bacteria in Tokyo, Sagami and Suruga Bays, respectively. Significant amounts of phosphatase-hydrolysable organic phosphorus were found in the euphotic zones of these bays and this organic phosphorus fraction accounted for 19 and 50% of organic phosphorus in Tokyo and Sagami Bays, respectively. Phosphatase-hydrolysable organic phosphorus was decomposed completely in the euphotic zone suggesting that this organic fraction is re-cycled in the primary production of the bays. Decomposition of natural organic phosphorus by enzymes was followed by measuring the release of inorganic orthophosphate from samples saturated with chloroform. Release of inorganic phosphorus proceeds rapidly for 2 or 3 days followed by a slow release. Enzymes present in the samples contributed to the decomposition of 50% of the organic phosphorus and the phosphatase enzyme was responsible for the decomposition of one-third of the hydrolysable organic phosphorus in the samples. Potential phosphatase activity in the samples was found to be indicative of the extent and rate of decomposition of organic phosphorus in coastal waters.     

Distribution and significance of heterotrophic marine bacteria with antibacterial activity.

Bacteria with antibacterial activity were isolated from seawater, sediments, phytoplankton, and zooplankton of Suruga, Sagami, and Tokyo Bays and from soft corals and sponges collected from the Taiwan coast. Of the 726 strains isolated, 37 showed antibacterial activity against either Vibrio parahaemolyticus (ATCC 17802) or Staphylococcus aureus (P209). Sediment harbored the lowest number of these forms of bacteria, and those from Tokyo Bay did not show any activity. Attached isolates showed greater activity compared with free-living forms. Relatively high numbers of strains with antibacterial activity were associated with phytoplankton. Among the zooplankton isolates, cladocerans harbored the maximum number of antibacterial strains. Isolates were more inhibitory to gram-positive test cultures. Autoinhibition was observed only among 8% of the isolates. Marine nonproducers were more susceptible. Pseudomonas/Alteromonas species made up 81.0% of isolates, of which 30% were pigmented strains. The absence or reduction in number of bacteria with antibacterial activity in Tokyo Bay is attributed to its eutrophic nature, which may tend to moderate the production of antibacterial compounds.     

Distribution and significance of heterotrophic marine bacteria with antibacterial activity

Bacteria with antibacterial activity were isolated from seawater, sediments, phytoplankton, and zooplankton of Suruga, Sagami, and Tokyo Bays and from soft corals and sponges collected from the Taiwan coast. Of the 726 strains isolated, 37 showed antibacterial activity against either Vibrio parahaemolyticus (ATCC 17802) or Staphylococcus aureus (P209). Sediment harbored the lowest number of these forms of bacteria, and those from Tokyo Bay did not show any activity. Attached isolates showed greater activity compared with free-living forms. Relatively high numbers of strains with antibacterial activity were associated with phytoplankton. Among the zooplankton isolates, cladocerans harbored the maximum number of antibacterial strains. Isolates were more inhibitory to gram-positive test cultures. Autoinhibition was observed only among 8% of the isolates. Marine nonproducers were more susceptible. Pseudomonas/Alteromonas species made up 81.0% of isolates, of which 30% were pigmented strains. The absence or reduction in number of bacteria with antibacterial activity in Tokyo Bay is attributed to its eutrophic nature, which may tend to moderate the production of antibacterial compounds.     

Some aspects of the biology of the goblin shark, <Emphasis Type="Italic">Mitsukurina owstoni</Emphasis>, collected from the Tokyo Submarine Canyon and adjacent waters,Japan

A total of 148 specimens of Mitsukurina owstoni were examined for morphology, distribution, stomach contents, and reproductive condition. Sixty-nine males (81.7–208.5 cm in total length, TL) and 56 females (92.8–196.1 cm TL) were collected with bottom gillnets along a steep slope of the Tokyo Submarine Canyon (100–350 m depth) from May 1995 to October 1996. In addition, data were obtained from 23 specimens of M. owstoni collected from Sagami Bay, Suruga Bay, and the Enshu-nada Sea from 1972 to 2001. Japanese catch records already published (from 1898) plus those in the present study were from the Pacific coast of Japan, from Choshi to the Kii Peninsula. The main fishing season for M. owstoni by bottom gillnet fishing in the Tokyo Submarine Canyon was October to April, at depths of 200–245 m. No specimens examined were sexually mature, including the largest male (208.5 cm TL) and female (196.1 cm TL) recorded. Of 110 stomachs from specimens from the Tokyo Submarine Canyon, 32 (29.1%) were empty. The remainder variously included teleost fishes, squids, decapods, isopods, digested food, and human refuse. Teleost fishes (Macrouridae sp. and Stomiidae spp.), an isopod (Cymothoidae), and decapods (Pasiphaea sinensis and Sergia sp.) were the only identifiable prey, the indication being that teleosts (all size ranges) were of primary importance as prey for M. owstoni.     

Aquatic environments: A potential source of antimicrobial-resistant Vibrio spp.

Vibrio spp. are associated with water and seafood-related outbreaks worldwide. They are naturally present in aquatic environments such as seawater, brackish water and freshwater environments. These aquatic environments serve as the main reservoirs of antimicrobial-resistant genes and promote the transfer of antimicrobial-resistant bacterial species to aquatic animals and humans through the aquatic food chain. Vibrio spp. are known as etiological agents of cholera and non-cholera Vibrio infections in humans and animals. Antimicrobial-resistant Vibrio species have become a huge threat in regard to treating Vibrio infections in aquaculture and public health. Most of the Vibrio spp. possess resistance towards the commonly used antimicrobials, including β-lactams, aminoglycosides, tetracyclines, sulphonamides, quinolones and macrolides. The aim of this review is to summarize the antimicrobial resistance properties of Vibrio spp. isolated from aquatic environments to provide awareness about potential health risks related to Vibrio infections in aquaculture and public health.     

Benthic ecology of <Emphasis Type="Italic">Vibrio</Emphasis> spp. and pathogenic <Emphasis Type="Italic">Vibrio</Emphasis> species in a coastal Mediterranean environment (La Spezia Gulf,Italy)

We carried out a 16-month in situ study to investigate the ecology of Vibrio spp. and pathogenic Vibrio species in coastal sediments of the Mediterranean Sea, employing multiple-regression analysis to reveal the major environmental factors controlling their occurrence in the benthic environment. In addition, association between vibrios and sediment-inhabiting meiofauna, which is a major component of benthic ecosystems, was investigated. Culturable and total Vibrio spp. estimates by most-probable-number technique coupled with standard polymerase chain reaction (PCR) and real-time PCR methods, respectively, were at least one order of magnitude higher in sediment than in seawater. In addition, potential human pathogenic species Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus occurred in the sediment with V. parahaemolyticus being the most frequently found. In the pelagic environment, 60% of total variance in culturable Vibrio data was explained by sea surface temperature (40%), salinity (13%) and organic matter concentration (7%). In the benthic environment, sea surface temperature was the only factor that significantly affected culturable Vibrio occurrence although it explained only 25% of total variance, suggesting that additional unexplored factors may play a role as well. No correlation was found between culturable Vibrio spp. concentrations and the abundance of harpacticoid copepods in the sediment whilst a negative correlation was found between Vibrio spp. and nematode abundance which accounted for almost 90% of the total meiofaunal density. Taxonomic analysis revealed that selective bacterial feeders accounted for nearly 50% of the total nematode community and included genera such as Terschellingia, Molgolaimus and Halalaimus, suggesting that top-down control by nematode grazing may be an important factor affecting Vibrio occurrence in these sediments. It is concluded that the benthic marine environment may function as a reservoir of Vibrio spp. and potential pathogenic vibrios whose ecological features appeared substantially different from the ones recognised in the pelagic environment.     

The in vitro antibiofilm activity of selected marine bacterial culture supernatants against Vibrio spp.

The aim of the work is to investigate the effect of marine bacterial culture supernatants on biofilm formation of Vibrio spp., a major menace in aquaculture industries. Vibrio spp. biofilm cause life-threatening infections in humans and animals. Forty-three marine bacterial culture supernatants were screened against the hydrophobicity index, initial attachment and biofilm formation in Vibrio spp. Twelve culture supernatants showed antibiofilm activity. The bacterial culture supernatants S8-07 (Bacillus pumilus) and S6-01 (B. indicus) inhibited the initial attachment, biofilm formation and dispersed the mature biofilm at 5% v/v concentration without inhibiting the growth. Analysis by light microscopy and confocal laser scanning microscopy showed that the architecture of the biofilm was destroyed by bacterial supernatants when compared to the control. The bacterial supernatants also reduce the surface hydrophobicity of Vibrio spp. which is one of the important requirements for biofilm formation. Further characterization of antibiofilm activity in S8-07 culture supernatant confirmed that it is an enzymatic activity and the size is more than 10 kDa and in S6-01, it is a heat-stable, non-protein compound. Furthermore, both the supernatants failed to show any biosurfactant activity. The culture supernatants of S8-07 and S6-01 with promising antibiofilm property have potential for application in medicine and marine aquaculture.     

Diving into the unknown: identification of antimicrobial resistance hotspots in a tropical urban estuary

Antimicrobial resistance is widely studied and well-characterized from a clinical perspective. However, considerably less information is available regarding resistance in environmental settings, especially in aquatic habitats. This study presents data regarding the occurrence, distribution and the antimicrobial susceptibility profile of bacteria isolated from Guanabara Bay (GB), a heavily polluted tropical urban estuary and an important tourist attraction in Rio de Janeiro, Brazil. Water samples from sites characterized by growing degrees of pollution were analysed by culture-dependent methods, revealing the presence of multidrug-resistant bacteria and clinically relevant indicators of antimicrobial resistance, such as extended-spectrum beta-lactamases. Isolates were identified by mass spectrometry, which indicated the presence of potential human pathogens such as Aeromonas spp. and Vibrio spp. Bacteria harbouring beta-lactam resistance genes were also detected. Although GB is widely used as a recreational and fishing area, there is a substantial knowledge gap regarding the monitoring of antimicrobial resistance and the risk that exposure to these waters poses to public health. Thus, this study reveals new information that calls for better comprehension of antimicrobial resistance in aquatic environments, especially those used for recreational purposes.     

Response of Bdellovibrio and Like Organisms (BALOs) to the Migration of Naturally Occurring Bacteria to Chemoattractants

A dual culture-based and non–culture-based approach was applied to characterize predator bacterial groups in surface water samples collected from Apalachicola Bay, Florida. Chemotaxis drop assays were performed on concentrated samples in an effort to isolate predator bacteria by their chemotactic ability. Yeast extract (YE) and casamino acids (CA) proved to be strong chemoattractants and resulted in three visibly distinct bands; however, dextrose, succinate, pyruvate, and concentrated cells of Vibrio parahaemolyticus P5 as prey did not elicit any response. The three distinct bands from YE and CA were separately collected to identify the chemotactic microbial assemblages. Plaque-forming unit assays from different chemotaxis bands with P5 as prey indicated 5- (CA) to 10-fold (YE) higher numbers of predator bacteria in the outermost chemotactic bands. Polymerase chain reaction–restriction fragment length polymorphism and 16S rDNA sequencing of clones from different chemotaxis bands resulted in identification of Pseudoalteromonas spp., Marinomonas spp., and Vibrio spp., with their numbers inversely proportional to the numbers of predators—i.e., Bdellovibrio spp. and Bacteriovorax spp—in the chemotaxis bands. This study indicates that predatorial bacteria potentially respond to high densities of microbial biomass in aquatic ecosystems and that chemotaxis drop assay may be an alternate culture-independent method to characterize predatorial bacterial guilds from the environment.     

Bacterial Growth on Coral Mucus

Coral mucus-degrading bacteria were isolated by an enrichment culture procedure. The isolates were able to grow as pure cultures on 10% sterilized mucus in seawater, yielding 10⁸ CFU/ml. The isolates, mostly Vibrio strains, were classified by classical and molecular methods. When carbon, nitrogen, and phosphorus compounds were added separately to the mucus medium, there was no increase in CFUs; however, when they were added together, there was a large increase in cell yield. The indigenous bacterial population of coral mucus increased from 10³ to 10⁸ CFU/ml when incubated at 30°C for 11 h, changing from a heterogeneous community to a Vibrio-dominated population. Factors which regulate the abundance and diversity of coral mucus bacteria are discussed.     

Differences in microbial communities between healthy and bleached coral Acropora solitaryensis from Xisha Islands,South China Sea

Serious bleaching events have been observed in Acropora solitaryensis, one of the main species of reef-building coral in the area of the Xisha islands in the South China Sea, during 2008–2011. The microbial communities of healthy and bleached coral samples were compared to explore the difference in the bacterial composition using 16S rRNA gene sequencing and denaturing gradient gel electrophoresis (DGGE). Analysis of 16S rRNA gene sequences showed that the diversity of bacteria from the corals was different between the healthy and the bleached. Albeit both the healthy coral and bleached coral displayed similar dominant bacterial species (α- and γ-proteobacteria), the ratio of Vibrio spp. increased sharply in the latter. As the bleaching developed, the diversity of the microbial community was dramatically decreased and the dominant species were replaced by γ-proteobacteria where Vibrio spp. and Escherichia spp. overwhelmed other genera. Similar results were gained by the DGGE technique though the abundance was lower. Furthermore, in the bleached tissues, Vibrio coralliilyticus was identified using both methods. These results indicated that pathogenic Vibrio spp. might be one of the factors causing A. solitaryensis bleaching in the coral reef of the Xisha islands.     

Method for estimating the decomposition of hexadecane in the marine environment.

A method, based on quantitating 14CO2 produced from [14C]hexadecane, has been developed for estimating the rate of hexadecane decomposition in seawater of Tokyo Bay during the summer stagnation period. The rate of hexadecane decomposition was from 0.1 to 1.3 mug/h per liter of seawater at the surface layer in the polluted gyre of the inner part of Tokyo Bay during the summer of 1974. A similar horizontal distribution pattern was seen for the density of hexadecane-decomposing bacteria.     

Inference of Potential Genetic Risks Associated with Large-Scale Releases of Red Sea Bream in Kanagawa Prefecture, Japan Based on Nuclear and Mitochondrial DNA Analysis

Since 1978, millions of hatchery-reared red sea bream (Pagrus major) juveniles have been released in Sagami Bay and Tokyo Bay in Kanagawa Prefecture, Japan. The stock enhancement program has contributed to total catch; however, no information regarding the genetic interactions with wild counterparts is available. Here, we combined 15 microsatellite loci and mitochondrial D-loop sequencing to characterize the genetic resources of red sea bream in Sagami Bay and Tokyo Bay and to elucidate the potential harmful genetic effects associated with fish releases. Both types of markers evidenced higher levels of genetic diversity in wild samples (SB and TB) compared with offspring before stocking (H07 and H08) as well as a hatchery-released sample recaptured in Sagami Bay (HR). Microsatellite F _ST estimates and Bayesian clustering analysis found significant genetic differences among samples (F _ST?=?0.013–0.054), except for the two wild samples (F _ST?=?0.002) and HR vs. H07 (F _ST?=?0.007). On the other hand, mitochondrial-based Ф _ST suggested haplotypic similarity between SB, H07, and HR. The low effective number of females contributing to the offspring over multiple generations may be responsible for the lack of haplotypic differentiation. Moreover, the putative hatchery origin to three fish (8 %) without deformity in the inter-nostril epidermis was inferred for the first time. Our results showed the usefulness of combining nuclear and mitochondrial markers to elucidate genetic interactions between hatchery-released and wild red sea bream and warned about potential harmful genetic effects should interbreeding takes place.     

Integrated Evaluation of Environmental Parameters Influencing <Emphasis Type="Italic">Vibrio</Emphasis> Occurrence in the Coastal Northern Adriatic Sea (Italy) Facing the Venetian Lagoon

In the marine environment, the persistence and abundance of Vibrio are related to a number of environmental parameters. The influence of the different environmental variables in determining the Vibrio occurrence could be different in the specific geographic areas around the world. Moreover, oceanographic parameters are generally interdependent and should not be considered separately when their influence on bacterial presence and concentration is tested. In this study, an integrated approach was used to identify key parameters determining the abundance of Vibrio spp in marine samples from the Venetian Lagoon in Italy, which is an important area for fish farming and tourism. Multivariate techniques have been adopted to analyze the dataset: using PCA, it was shown that a relatively high proportion of the total variance in this area was mainly due to two independent variables, namely salinity and temperature. Using cluster analysis, it was possible to categorize different groups with homogeneous features as regards space (“stations”) and time (“seasons”) distribution, as well as to quantify the values of environmental variables and the Vibrio abundances in each category. Furthermore, integrating key environmental factors and bacterial concentration values, it was possible to identify levels of salinity and sea surface temperature which were optimal for Vibrio concentration in water, plankton, and sediment samples. The identification of key environmental variables conditioning Vibrio occurrence should facilitate ocean monitoring, making it possible to predict unexpected variations in marine microflora which determine possible public health risks in coastal areas.     

培养法检测海滨浴场细菌多样性及耐药性研究

【目的】耐药基因细菌水平基因转移导致的耐药菌数量增加引发的公共安全问题日益引起人们关注,监测环境中耐药菌变得极为重要。【方法】采集湛江3处滨海浴场的水体、沙滩土样,通过平板稀释涂布和琼脂扩散法进行浴场微生物数量、多样性和抗生素耐药性分析。【结果】3处浴场水体无机氮含量偏高,浴场微生物数量随着客流量逐渐增加,沙滩中微生物数量显著高于水体。浴场细菌分布于3门12科18属,水体中变形菌门（Proteobacteria,49.64%）占优势,沙滩则是厚壁菌门（Firmicutes,54.74%）占优势。浴场细菌对β-内酰胺类耐药率较高,青霉素、万古霉素和头孢曲松耐药率分别达到23.25%、20.53%和17.42%,耐药菌株主要分布于芽孢杆菌属（Bacillus）、弧菌属（Vibrio）、假单胞菌属（Pseudomonas）、链霉菌属（Streptomyces）和肠杆菌属（Enterobacter）,水体中多重耐药细菌数量显著高于沙滩,集中于人流量多的浴场。【结论】滨海浴场环境中细菌耐药菌种类多,需持续监测以评估对当前地区公共卫生的潜在影响。     

Biochemical characteristics and genetic diversity of <Emphasis Type="Italic">Vibrio</Emphasis> spp. and <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> strains isolated from the Lac of Bizerte (Tunisia)

This study characterized the phenotypic and genetic properties of Vibrio spp. and Aeromonas hydrophila strains isolated from seawater and mussels (Mytilus edulis and Crassostrea gigas) cultured in mollusc farm localized in the lac of Bizerte. The 37 strains (31 strains of V. alginolyticus, one strain of V. fluvialis, one strain of V. parahaemolyticus and four strains of A. hydrophila) typed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) showed a high polymorphism. Most of the isolates were resistant to at least two antimicrobial agents. All the tested strains were resistant to ampicillin. PCR was used to detect the presence of eight Vibrio cholerae virulence genes in the genome of the Vibrio spp. isolates. The results showed a wide dissemination of these genes in the genome of all Vibrio spp. isolates tested. Differentiation of these strains with the ERIC 2-PCR technique revealed no association between the presence of virulence genes and a particular fingerprinting pattern.     

Bacterial composition of sponges,sediment and seawater in enclosed and open marine lakes in Ha Long Bay Vietnam

ABSTRACT

Marine lakes are landlocked bodies of seawater, isolated to varying degrees from the surrounding marine habitat. Isolated lakes generally have lower pH values, salinities and higher temperatures than more open lakes. We used a 16S rRNA gene barcoded pyrosequencing approach to study the bacterial communities of two sponge species, sediment and seawater in one enclosed and two open marine lakes. Bacterial communities of the sponge Spheciospongia solida mainly consisted of Proteobacteria, Cyanobacteria and Bacteroidetes. In contrast, Proteobacteria, Chloroflexi and Acidobacteria dominated the bacterial communities of the sponge Spongia ceylonensis. Although only a limited amount of samples were collected, both water and S. ceylonensis sponge had higher relative abundances of Cyanobacteria in the enclosed lake, which mainly consisted of OTUs assigned to the genus Synechococcus. This is in line with a number of previous studies, which have shown that environmental conditions found within low pH environments such as marine lakes benefit the growth of Synechococcus spp. Future studies should address the mechanism by which Synechococcus spp. may help host sponges and their bacterial communities adapt to low pH conditions in isolated marine lakes and other low-pH environments.     

Vibrio infections triggering mass mortality events in a warming Mediterranean Sea

Mass mortality events of benthic invertebrates in the temperate north‐western (NW) Mediterranean Sea have been observed in recent seasons. A 16 month in situ study in the Ligurian Sea (NW Mediterranean Sea) demonstrated that the occurrence of Paramuricea clavata mortality episodes were concomitant to a condition of prolonged high sea surface temperatures, low chlorophyll concentrations and the presence of culturable Vibrio spp. in seawater. The occurrence of Vibrio spp. at the seasonal scale was correlated with temperature; with few vibrios retrieved on specific media when the temperature dropped below 18°C and a sharp increase of vibrios abundance (up to 3.4 × 10⁴ MPN l^?1) when the temperature was greater than or equal to 22°C. Phylogenetic and phenotypic analysis of Vibrio isolates associated with healthy and diseased P. clavata colonies collected during a mortality episode showed that these bacteria were significantly more abundant in diseased than in healthy corals and were related to the V. harveyi, V. splendidus and V. coralliilyticus groups, the latter only identified in diseased organisms. Inoculation of bacterial isolates from these groups onto healthy P. clavata in aquaria caused disease signs and death in a range of Vibrio concentrations, temperature values and trophic conditions consistent with those recorded in the field. It is concluded that Vibrio infections may act as an additional triggering mechanism of mass mortality events in the coastal Mediterranean Sea and that their occurrence is climate‐linked. Predicted global warming leading to long‐lasting hot summer periods together with stratification resulting in energetic constraints represent a major threat to the survival of benthic invertebrates in the temperate NW Mediterranean Sea due to potential disease outbreak associated with Vibrio pathogens.     

Agar Plate Method for Detection and Enumeration of Alkylbenzenesulfonate-Degrading Microorganisms

A simple method for detection and enumeration of alkylbenzenesulfonate (ABS)-degrading microorganisms by using agar plates was developed and used in microbiological studies of coastal marine and polluted river waters. The method depends upon the color responses of neutral red in alkaline medium. Neutral red changes from pink, when it enters into ABS micelles, to yellow, when the ABS is degraded, and does not form micelles. When neutral red-tris(hydroxymethyl)-aminomethane buffer solution and then cationic surfactant solution were sprayed onto the agar surface of ABS-nutrient agar cultures, transparent haloes appeared around the colonies of ABS-degrading microorganisms against a pink background. Viable counts of ABS-degrading bacteria isolated from both seawater and freshwater environments were considerably higher in polluted waters than in less polluted areas. Viable counts of ABS-degrading bacteria averaged 1.5 x 105/ml in samples from the surface water of polluted Tokyo Bay and 3.0 x 104/ml in samples from the surface water of polluted Tamagawa River but were fewer in number in samples from less polluted waters.     

Vibrio diseases of marine fish populations

SeveralVibrio spp. cause disease in marine fish populations, both wild and cultured. The most common disease, vibriosis, is caused byV. anguillarum. However, increase in the intensity of mariculture, combined with continuing improvements in bacterial systematics, expands the list ofVibrio spp. that cause fish disease. The bacterial pathogens, species of fish affected, virulence mechanisms, and disease treatment and prevention are included as topics of emphasis in this review.