Discovery of a Marine Bacterium Producing 4-Hydroxybenzoate and Its Alkyl Esters, Parabens

Chemically synthesized 4-hydroxybenzoate (4HBA) is widely used in the chemical and electrical industries as a material for producing polymers such as those of the liquid crystal type. Its alkyl esters, called parabens, have been the most widely used preservatives by the food and cosmetic industries. We report here for the first time a microorganism, a marine bacterium, which biosynthesizes these petrochemical products. The marine bacterial strain, A4B-17, which was found to belong to the genus Microbulbifer on the basis of its rRNA and gyrB sequences, was isolated from an ascidian in the coastal waters of Palau. Strain A4B-17 was, surprisingly, found to produce 10 mg/liter of 4HBA, together with its butyl (24 mg/liter), heptyl (0.4 mg/liter), and nonyl (6 mg/liter) esters. We therefore characterized 23 other marine bacteria belonging to the genus Microbulbifer, which our institute had previously isolated from various marine environments, and found that these bacteria also produced 4HBA, although with low production levels (less than one-fifth of that produced by A4B-17). We also show that the alkyl esters of 4HBA produced by strain A4B-17 were effective in preventing the growth of yeasts, molds, and gram-positive bacteria.     

Experimentally induced marine flexibacteriosis in Atlantic salmon smolts Salmo salar. I. Pathogenicity

Tenacibaculum maritimum causes marine flexibacteriosis in many cultured fish species, including Atlantic salmon Salmo salar in Tasmania, Australia. Several aspects of the pathogenicity of this bacterium were investigated in naive Atlantic salmon smolts using different isolates, growth conditions and doses to produce a model of infection. We found that T. maritimum is pathogenic to Atlantic salmon using either marine Shieh's or marine Ordal's culture medium. The use of aeration in broth culture produced a dose effect in challenge due to a 'clumping' of the bacteria during culture. The virulence of a strain appears to be connected with this 'clumping'; the more adherent the cells, the more pathogenic the strain. Differences in virulence between 3 strains was apparent, with 1 of the strains (89/4747) being non-pathogenic and unable to produce disease in the host. The 2 other strains (89/4762, 00/3280) were highly virulent, resulting in 100% mortalities within 3 d. A reproducible model of infection has been established in the present study using strain 89/4762. Results from the present study provide a better insight into the nature of the disease.     

Antimicrobial activity of surface attached marine bacteria in biofilms

Relatively little is known about the microbial ecology of biofilm communities or the diversity of antimicrobial molecules that they produce to regulate these communities. This study tested whether the production of antimicrobial activity in biofilm cultures is enhanced towards competing bacteria found in those biofilms. First, the production of antimicrobial activity of marine bacteria grown in biofilms was tested. Fourteen of the 105 marine isolates tested were found to produce antimicrobial factors when grown in biofilms. The antimicrobial activity produced by these isolates in biofilms was more potent and inhibited a broader range of target bacteria grown in biofilms compared to shaken liquid cultures. In a separate experiment, we found that cultivation in biofilms containing produced metabolites from an ‘inducer’ bacterium stimulated the production of antimicrobial molecules by ‘producer’ bacteria that were active against the ‘inducer’ bacterium. Overall, the study suggests that surface attached marine bacteria can target their antimicrobial activity towards competing bacteria in biofilms.     

Antarctic microorganisms as source of the omega-3 polyunsaturated fatty acids

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are long-chain polyunsaturated fatty acids (PUFAs) that belong to the omega-3 group. They are essential fatty acids found in phospholipid of cell membranes. There is strong evidence that these nutrients may also favorably modulate many diseases. Primary sources of omega-3 PUFAs in the human diet are fish and fish-derived products. The fishing industry worldwide, however, is becoming unable to satisfy the growing demand for these PUFAs. A promising cost-effective alternative source of PUFAs is bacterial production. We identified 40 Antarctic marine bacterial isolates by 16S rRNA gene sequence analysis. Fifteen genera in three phyla were represented in the collection. Isolates were tested for ability to produce EPA using a method in which their ability to reduce 2,3,5-triphenyltetrazolium chloride (TTC) is determined and by gas chromatography coupled to mass spectrometry (GC–MS). All isolates could reduce TTC, and GC–MS analysis showed that four produced EPA and that six produced DHA. We show for the first time that isolates identified as Cellulophaga, Pibocella and Polaribacter can produce EPA and DHA, only DHA or only EPA, respectively. One isolate, Shewanella sp. (strain 8-5), is indicated to be a good candidate for further study to optimize growth and EPA production. In conclusion, a rapid method was tested for identification of new EPA producing strains from marine environments. New EPA and DHA producing strains were found as well as a potentially useful PUFA production strain.     

Discovery of a marine bacterium producing 4-hydroxybenzoate and its alkyl esters, parabens

Chemically synthesized 4-hydroxybenzoate (4HBA) is widely used in the chemical and electrical industries as a material for producing polymers such as those of the liquid crystal type. Its alkyl esters, called parabens, have been the most widely used preservatives by the food and cosmetic industries. We report here for the first time a microorganism, a marine bacterium, which biosynthesizes these petrochemical products. The marine bacterial strain, A4B-17, which was found to belong to the genus Microbulbifer on the basis of its rRNA and gyrB sequences, was isolated from an ascidian in the coastal waters of Palau. Strain A4B-17 was, surprisingly, found to produce 10 mg/liter of 4HBA, together with its butyl (24 mg/liter), heptyl (0.4 mg/liter), and nonyl (6 mg/liter) esters. We therefore characterized 23 other marine bacteria belonging to the genus Microbulbifer, which our institute had previously isolated from various marine environments, and found that these bacteria also produced 4HBA, although with low production levels (less than one-fifth of that produced by A4B-17). We also show that the alkyl esters of 4HBA produced by strain A4B-17 were effective in preventing the growth of yeasts, molds, and gram-positive bacteria.     

Quorum sensing signals are produced by Aeromonas salmonicida and quorum sensing inhibitors can reduce production of a potential virulence factor

Many pathogens control production of virulence factors by self-produced signals in a process called quorum sensing (QS). We demonstrate that acyl homoserine lactone (AHL) signals, which enable bacteria to express certain phenotypes in relation to cell density, are produced by a wide spectrum of Aeromonas salmonicida strains. All 31 typical strains were AHL producers as were 21 of 26 atypical strains, but on a strain population basis, production of virulence factors such as protease, lipase, A-layer or pigment did not correlate with the production and accumulation of AHLs in the growth medium. Pigment production was only observed in broth under highly aerated conditions. Quorum sensing inhibitors (QSIs) are compounds that specifically block QS systems without affecting bacterial growth and 2 such compounds, sulphur-containing AHL-analogues, reduced production of protease in a typical strain of Aeromonas salmonicida. The most efficient compound N-(heptylsulfanylacetyl)-L-homoserine lactone (HepS-AHL), reduced protease production by a factor of 10. Five extracellular proteases were detected on gelatin-containing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels and 3 of these were completely down regulated by HepS-AHL. Hence, QSIs can curb virulence in some strains and could potentially be pursued as bacterial disease control measures in aquaculture.     

Description of a bacteriocinogenic plasmid in Beneckea harveyi.

A total of 795 strains of marine Vibrio species and Beneckea harveyi, a luminescent marine bacterium, were isolated from various sources in the area of Galveston Island, Tex., and screened for the production of bacteriocin-like substances. More than 8% of the Vibrio isolates produced low-molecular-weight (dialyzable) substances, which were lethal to a test strain of V. parahaemolyticus. Approximately 5% of the B. harveyi isolates produced higher-molecular-weight (nondialyzable) substances which were lethal to a test strain of B. harveyi. One of the B. harveyi strains (strain SY) produced a nondialyzable substance which was lethal to two of 39 strains of B. harveyi. The substance showed no activity toward 17 test strains drawn from the Vibrionaceae and Enterobacteriaceae. Strain SY showed no sensitivity to its own lethal agent and was shown by agarose gel electrophoresis and electron microscopy to harbor a single plasmid of 38 x 10(6) daltons. Variants of strain SY lacking the plasmid were produced by growth in the presence of the antibiotic novobiocin. These variants lacked both the ability to produce the lethal substance and the ability to survive in its presence. The lethal agent produced by strain SY is the first bacteriocin reported in marine bacteria. The term "harveyicin" is proposed to name this lethal substance.     

Enzyme inhibitors and other bioactive compounds from marine actinomycetes

Several enzyme-inhibitor-producing actinomycetes were isolated from various samples collected from the marine environment and characterized. Most of them produced novel compounds that are useful in medicine and agriculture. Actinomycete strain no. 18, which produces antibiotics against Gram-positive bacteria only in the presence of seawater, was isolated from sediment sampled from neritic sea water and characterized. The production of antibiotics was observed at seawater concentrations ranging from 60 to 110% (v/v). Thus, the production was seawater-dependent. The production of tetrodotoxin (TTX), known otherwise as puffer fish toxin, was investigated in various actinomycetes collected from the marine environment. Of 10 isolates from various sea areas, 9 produced TTX as judged by their retention times on high-performance liquid chromatography (HPLC). To our knowledge, this is the first report of actinomycetes from the marine environment that produce TTX.     

海洋链霉菌GB-2发酵产物的抗细菌活性及性质研究

从连云港海域潮间带采集的样品中筛选得到一株产高活性抗细菌物质的链霉菌GB-2。该菌的发酵产物对蜡样芽孢杆菌AS1.1846、金黄色葡萄球菌ATCC25923及6株耐药性金黄色葡萄球菌等11株革兰氏阳性菌,大肠杆菌AS1.487、荧光假单胞菌AS1.1802等4株革兰氏阴性菌有显著拮抗作用。纸层析对抗细菌物质分析结果表明,菌株GB-2所产抗细菌物质是中性的水溶性物质,其产生与海水的存在有显著相关性。发酵液稳定性研究表明,该物质在121℃,pH1和pH12条件下抑菌活性均不变;紫外线照射也不影响其抑细菌活性。显示菌株GB-2产物在生防、食品及医药方面潜在的应用价值。     

Hydroxamate-siderophore production and utilization by marine eubacteria

Siderophore (iron-binding chelator) production was examined in 30 strains of open ocean bacteria from the generaVibrio, Alteromonas, Alcaligenes, Pseudomonas, andPhotobacterium. The results showed that hydroxamate-type siderophore production was widely distributed in various marine species, except for isolates ofAlteromonas macleodii andV. nereis. In all cases, the ability to produce siderophores was under the control of iron levels in the medium and satisfied the iron requirements of the siderophore bioassay organism. On the basis of chemical assay and bacterial bioassays, none of the examined isolates produced phenolate-type siderophores. Several isolates produces siderophores that were neither hydroxamatenor phenolate-type siderophores. Some strains such asAlteromonas communis produce siderophores that could be used by many other isolates. In contrast, the siderophore produced byAlcaligenes venustus had little cross-strain utilization. These findings suggest that the ability to produce siderophores may be common to open ocean bacteria.     

Competitive interactions in mixed-species biofilms containing the marine bacterium Pseudoalteromonas tunicata

Pseudoalteromonas tunicata is a biofilm-forming marine bacterium that is often found in association with the surface of eukaryotic organisms. It produces a range of extracellular inhibitory compounds, including an antibacterial protein (AlpP) thought to be beneficial for P. tunicata during competition for space and nutrients on surfaces. As part of our studies on the interactions between P. tunicata and the epiphytic bacterial community on the marine plant Ulva lactuca, we investigated the hypothesis that P. tunicata is a superior competitor compared with other bacteria isolated from the plant. A number of U. lactuca bacterial isolates were (i) identified by 16S rRNA gene sequencing, (ii) characterized for the production of or sensitivity to extracellular antibacterial proteins, and (iii) labeled with a fluorescent color tag (either the red fluorescent protein DsRed or green fluorescent protein). We then grew single- and mixed-species bacterial biofilms containing P. tunicata in glass flow cell reactors. In pure culture, all the marine isolates formed biofilms containing microcolony structures within 72 h. However, in mixed-species biofilms, P. tunicata removed the competing strain unless its competitor was relatively insensitive to AlpP (Pseudoalteromonas gracilis) or produced strong inhibitory activity against P. tunicata (Roseobacter gallaeciensis). Moreover, biofilm studies conducted with an AlpP- mutant of P. tunicata indicated that the mutant was less competitive when it was introduced into preestablished biofilms, suggesting that AlpP has a role during competitive biofilm formation. When single-species biofilms were allowed to form microcolonies before the introduction of a competitor, these microcolonies coexisted with P. tunicata for extended periods of time before they were removed. Two marine bacteria (R. gallaeciensis and P. tunicata) were superior competitors in this study. Our data suggest that this dominance can be attributed to the ability of these organisms to rapidly form microcolonies and their ability to produce extracellular antibacterial compounds.     

Biosynthesis of bile acids in a variety of marine bacterial taxa

Several marine bacterial strains, which were isolated from seawater off the island Dokdo, Korea, were screened to find new bioactive compounds such as antibiotics. Among them, Donghaeana dokdonensis strain DSW-6 was found to produce antibacterial agents, and the agents were then purified and analyzed by LC-MS/MS and 1D- and 2D-NMR spectrometries. The bioactive compounds were successfully identified as cholic acid and glycine-conjugated glycocholic acid, the 7alpha-dehydroxylated derivatives (deoxycholic acid and glycodeoxycholic acid) of which were also detected in relatively small amounts. Other masine isolates, taxonomically different from DSW-6, were also able to produce the compounds in a quite different production ratio from DSW-6. As far as we are aware of, these bile acids are produced by specific members of the genus Streptomyces and Myroides, and thought to be general secondary metabolites produced by a variety of bacterial taxa that are widely distributed in the sea.     

Studies in the differentiation between Microsporum ferrugineum Ota and Trichophyton soudanense Joyeux

A study, conducted with 20 isolates of Microsporum ferrugineum and 12 isolates of Trichophyton soudanense, revealed that some of the discrepancies in the literature regarding their characteristics and differentiation were due to methodology, strain variation and the use of an insufficient number of isolates. We found all isolates of T. soudanense to be urease negative and gelatinase positive (usually by the first week); to produce brown to black colonies on Lowenstein-Jensen medium; to rapidly decompose casein and more slowly tyrosine; to grow well or better at 37°C as compared to room temperature; to produce reflexive branching on cornmeal Tween agar and abundant microconidia on casero medium and to exhibit no sexual reaction with either mating type of Arthroderma simii. All but one isolate demonstrated restricted growth on lactose agar and only three isolates perforated hair.In contrast, we found 18 of 20 isolates of M. ferrugineum to be urease positive in urea broth (most isolates were negative on urea agar); all produced light-colored colonies on Lowenstein-Jensen medium; spreading colonies on lactose agar and failed to perforate hair in vitro or to produce reflexive branching. Most isolates manifested poorer to no growth at 37°C compared to room temperature and all but one failed to decompose casein and tyrosine. A few strains produced macroconidia and/ or microconidia on casero medium and some reacted sexually with A. simii (a) or (–) mating type. Gelatin hydrolysis was variable.We suggest the following key tests to differentiate M. ferrugineum from T. soudanense: urease activity in urea broth; colony color on Lowenstein-Jensen medium; growth on lactose agar; growth at 37° C compared to room temperature; presence of reflexive branching on cornmeal Tween agar.     

Control of Listeria monocytogenes in a biofilm by competitive-exclusion microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37 degrees C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10(3) CFU of L. monocytogenes/ml and 10(5) CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37 degrees C for 24 h, 15 degrees C for 14 days, 8 degrees C for 21 days, and 4 degrees C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37 degrees C, two at 15 and 8 degrees C, and three at 4 degrees C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4 degrees C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log(10) CFU of L. monocytogenes/cm(2)). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37 degrees C.     

Chemical characterization and quantification of siderophores produced by marine and terrestrial aspergilli

Ten aspergilli (five each from marine and terrestrial habitats) were screened for siderophore production. All test isolates produced siderophores as indicated by a positive reaction in the FeCl(3) test, chrome azurol sulphonate assay, and chrome azurol sulphonate agar plate test. Further, the test isolates were compared for their siderophore production potential and chemical characteristics. Examination of the chemical nature of the siderophores revealed that all test isolates produced hydroxamate siderophores that were trihydroxamate hexadentates. Wide-spread occurrence of siderophores in marine isolates indicate their functional role in maintaining overall productivity of coastal waters. Among all test aspergilli, marine Aspergillus versicolor was found to be the largest siderophore producer (182.5 microg/mL desferrioxamine mesylate equivalent), least siderophore production was recorded in a marine strain of Aspergillus niger (3.5 microg/mL desferrioxamine mesylate equivalent).     

Antimicrobial activity observed among cultured marine epiphytic bacteria reflects their potential as a source of new drugs

The surfaces of marine eukaryotes provide a unique habitat for colonizing microorganisms where competition between members of these communities and chemically mediated interactions with their host are thought to influence both microbial diversity and function. For example, it is believed that marine eukaryotes may use their surface-associated bacteria to produce bioactive compounds in defence against competition and to protect the host against further colonization. With the increasing need for novel drug discovery, marine epibiotic bacteria may thus represent a largely underexplored source of new antimicrobial compounds. In the current study, 325 bacterial isolates were obtained from the surfaces of marine algae Delisea pulchra and Ulva australis . Thirty-nine showed to have antimicrobial activity and were identified via 16S rRNA gene sequencing. The majority of those isolates belonged to Alpha- and Gammaproteobacteria . Interestingly, the most commonly isolated bacterial strain, Microbulbifer sp., from the surface of D. pulchra has previously been described as an ecologically significant epibiont of different marine eukaryotes. Other antimicrobial isolates obtained in this study belonged to the phyla Actinobacteria , Firmicutes and Bacteroidetes . Phylogenetically, little overlap was observed among the bacteria obtained from surfaces of D. pulchra and U. australis . The high abundance of cultured isolates that produce antimicrobials suggest that culturing remains a powerful resource for exploring novel bioactives of bacterial origin.     

Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates.

One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.     

H107, a new aminoglycoside anti-Pseudomonas antibiotic produced by a new strain of Spirillospora

Spirillospora spp. (strain 719) has been the source of several antibiotics. One of these designated H107 is produced as a trace. Compared with other antibiotics produced by the same strain, it was obtained only from the broth filtrate after precipitation with acetic acid followed by extraction with n-butanol. It was a water soluble metabolite active against Gram-negative bacteria and especially Pseudomonas spp., and was identified as an aminoglycoside compound. This is the first report of aminoglycoside anti-Pseudomonas production by Spirillospora.     

Antagonistic activity against pathogenic bacteria by human vaginal lactobacilli

This study attempted to isolate lactobacilli strains from healthy vaginal ecosystem to search for a new effective antibacterial probiotic strain. The strains were identified and characterized for their probiotic properties including bile salt and acid tolerance, growth at acidic pH, their ability to utilize protein, starch, and lipid, the production of hydrogen peroxide and bacteriocin as well as their antibiotic resistance patterns. The antibacterial activity of the culture supernatant of these strains were tested against a wide range of Gram-positive and Gram-negative pathogenic bacteria including Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae. Salmonella typhi, and Salmonella typhimurium. None of the strains inhibited the growth of Gram-negative bacteria. Contrastly, the culture supernatant of strain L 22, identified as Lactobacillus reuteri, significantly inhibited all of the clinical isolates of methicillin-resistant S. aureus (MRSA). The antibacterial effect of the selected strain L 22 was further investigated. In the presence of L 22, the bacterial growth was assessed in vitro by viable bacterial counting. The numbers of viable cells were significantly lower in L 22-containing broth than those in the control by 6h. This finding clearly demonstrates that strain L 22 can produce an anti-MRSA effect. The antibacterial ability of the strain L 22 was fundamentally attributed to their bacteriocin production which can cause both cell inhibition and cell death.