Antiviral and immunomodulatory effects of a novel bacterial exopolysaccharide of shallow marine vent origin.

AIMS: To evaluate a novel exopolysaccharide (EPS1) from the recently described haloalkaliphilic, thermophilic Bacillus licheniformis strain T14, isolated from a shallow hydrothermal vent of Panarea Island (Italy), for its antiviral and immunomodulatory effects against herpes simplex virus type 2 (HSV-2). METHODS AND RESULTS: EPS1-T14 hindered the HSV-2 replication in human peripheral blood mononuclear cells (PBMC) but not in WISH cells, indicating that cell-mediated immunity was involved in the antiviral activity. High levels of Th1-type cytokines were detected in supernatants of EPS1-treated PBMC, while Th2-type cytokines were not induced. CONCLUSIONS: The novel EPS1-T14 is a water-soluble, noncytotoxic exopolymer able to stimulate the immune response and thus contribute to the antiviral immune defence, acting as immunomodulator. SIGNIFICANCE AND IMPACT OF THE STUDY: The exopolysaccharide produced by B. licheniformis strain T14, stimulator of Th1 cell-mediated immunity, could be used as therapy in immunocompromised host.

New bacilli from shallow hydrothermal vents of Panarea Island (Italy) and their biotechnological potential.

AIMS: To characterize bacilli isolated from shallow hydrothermal vents of Panarea Island (Italy) and evaluate their biotechnological potential. METHODS AND RESULTS: Fifteen isolates were characterized by culture and molecular methods. Eleven isolates were thermophilic, six isolates were alkalophilic and four of them were haloalkalophilic. After 16S rRNA gene sequencing, four strains, exhibiting sequence similarity below 95% with deposited strains, may represent novel species of bacilli. One strain was strictly related to Geobacillus subterraneus, but shared phenotypic characteristics for which it could be considered a new strain of this species. Four strains were affiliated with different Bacillus spp. Most isolates produced gelatinase, lipases and amylase, and some were mercury tolerant. Exopolysaccharides (EPS) production was tested adding different sugars (glucose, sucrose, trehalose, fructose, ribose, xylose and mannose, 1% w/v) as a carbon source in a minimal medium. The highest EPS yield (185 mg l(-1)) was reached by strain 1A70 utilizing ribose as a carbon source. CONCLUSIONS: Novel strains of Geobacillus and indigenous ribotypes of Bacillus with biotechnological potential inhabit shallow vents of Panarea Island. SIGNIFICANCE AND IMPACT OF THE STUDY: New strains of thermophilic bacilli from Panarea are producers of useful biomolecules for industrial purposes as well as environmental and biotechnological applications.

Conventional and molecular methods to detect bacterial pathogens in mussels.

AIM: To detect Aeromonas spp., Salmonella spp., Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus in mussels and water samples from a farming area, conventional and molecular methods were applied to enrichment cultures. METHODS AND RESULTS: The aerolysin gene (aero) of Aeromonas spp., the invasion plasmid antigen B (ipaB) gene of Salmonella spp., the enterotoxin secretion protein (epsM) gene of V. cholerae, the species-specific region of 16S rRNA gene of V. vulnificus, the 16S-23S rDNA (IGS) gene of V. parahaemolyticus and the pR72H fragment of V. parahaemolyticus were amplified by multiplex polymerase chain reaction (PCR) assays on DNA extracted from enrichment cultures. The haemolysin gene (tdh) of pathogenic V. parahaemolyticus was also amplified. Conventional culture method allowed the isolation of V. parahaemolyticus and V. vulnificus from water and mussels. The genes aero, epsM and 16S rRNA of V. vulnificus were occasionally detected in the enrichment cultures. In mussels, the ipaB and IGS genes were detected from June to September and from April to November, respectively. All genes, except aero, were amplified from mussels collected in September, when pathogenic V. parahaemolyticus (tdh+) strains were also isolated. CONCLUSIONS: Multiplex-PCR assays were more sensitive and faster than conventional procedures. SIGNIFICANCE AND IMPACT OF THE STUDY: The results emphasize the need of an accurate and rapid detection of bacterial pathogens in mussels to protect human health.

Specific detection of Arcobacter spp. in estuarine waters of Southern Italy by PCR and fluorescent in situ hybridization.

AIM: To evaluate the reliability of culture-independent methods in comparison with culture-dependent ones for the detection of Arcobacter spp. in estuarine waters of Southern Italy. METHODS AND RESULTS: PCR and fluorescent in situ hybridization (FISH) procedures were used to detect arcobacters directly in water samples and after enrichment cultures. The samples totally were positive by molecular methods (PCR and FISH) but only 75% were culture positive, confirming the limitation of these latter to detect Arcobacter spp. in natural samples. Culturable arcobacters were retrieved in all times except in July, and isolated species were ascribed only to Arcobacter cryaerophilus. CONCLUSIONS: Culturable and nonculturable forms of Arcobacter in the estuarine environment were present. PCR assays were more sensitive than traditional culture in detecting Arcobacter butzleri and A. cryaerophilus. FISH comparatively to PCR technique may provide information about cell morphology and viability of single cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Our investigation indicates the existence of an environmental reservoir of potential pathogenic arcobacters in an estuarine Italian area, which may survive under a viable but not culturable state.

Induction and resuscitation of viable nonculturable Arcobacter butzleri cells.

Two strains of Arcobacter butzleri, ATCC 49616 and an environmental isolate, became nonculturable in seawater microcosms at 4 degrees C by 20 days and at room temperature by 14 days. Nonculturable cells were viable for up to 270 days of incubation in microcosms. Resuscitation of A. butzleri cells from microcosms at both temperatures was achieved 9 days after nutrient addition.

Polyphasic approach to the characterisation of marine luminous bacteria.

Fifteen luminous bacterial strains were isolated from the Tyrrhenian Sea coastal waters off northeastern Sicily and characterised by a combination of phenotypic and molecular tests in order to identify them and to determine their intraspecific genetic variability. Five luminous type strains, Vibrio splendidus NCIMB 1, V. harveyi NCIMB 1280, V. fischeri NCIMB 1281, V. orientalis NCIMB 2195 and Photobacterium leiognathi NCIMB 2193, were used as reference. On the basis of their phenotypic characters, the isolates were assigned to the family Vibrionaceae and all were related to the V. harveyi reference strain. Amplified 16S ribosomal DNA restriction analysis (ARDRA) enabled the strains to be subdivided into three groups, two of which exhibited the same restriction pattern as the two reference strains, V. harveyi and V. splendidus. Comparison of the full 16S rDNA sequence and of a 100-bp highly variable 16S rDNA region (selected as a 'signature' sequence for the luminous bacteria) confirmed ARDRA data and suggested that the strains of the third group could be considered a subspecies of V. harveyi or a tyrrhenian biovar, different from the other reference strains whose 16S rDNAs have already been sequenced. Random amplified polymorphic DNA (RAPD) fingerprinting and analysis of plasmid content suggested a high degree of intraspecific genetic variability within the largest ARDRA group. Data obtained suggest that the ARDRA method and the sequencing of the 16S rDNA signature region could be a powerful tool for a rapid identification of marine luminous bacteria.

In vitro susceptibility of Arcobacter butzleri and Arcobacter cryaerophilus to different antimicrobial agents.

Seventeen strains of Arcobacter butzleri and thirteen of Arcobacter cryaerophilus, were tested for their antimicrobial susceptibility to 26 antimicrobial agents. Among beta-lactams agents in this study, imipenem was the most active agent against both A. butzleri and A. cryaerophilus isolates with MIC(90) values of 2 and 4 mg/l, respectively. The most active cephalosporin tested was cefepime, although it was more active against A. butzleri (MIC(90) 8 mg/l) than A. cryaerophilus (MIC(90) 64 mg/l). Levofloxacin, marbofloxacin, enrofloxacin and ciprofloxacin were the best-performing fluoroquinolones against these species. Of the aminoglycosides, amikacin was the most active agent against both A. butzleri and A. cryaerophilus strains with MIC(90) values of 64 and 16 mg/l, respectively. All isolates showed high levels of resistance to penicillins, macrolides, chloramphenicol, trimethoprim and vancomycin.

A polyphasic taxonomic study of thermophilic bacilli from shallow, marine vents.

Eighty-seven thermophilic, aerobic, spore-forming bacteria were isolated from shallow, marine, thermal vents of the Eolian Islands (Italy) and tested for a broad spectrum of phenotypic characteristics. A numerical taxonomy study was performed on these isolates and 8 thermophilic Bacillus and Geobacillus reference strains by 89 selected features. Results from cluster analysis showed the formation of nine clusters. Most of the isolates (83%) fell into several phenetically well distinguished clusters, loosely related to Geobacillus thermodenitrificans. The remaining isolates grouped together with different reference strains. Eighteen isolates, representative of the different clusters, were selected for subsequent genotypic characterisation, including partial 16S rDNA sequence analysis of 18 strains and almost complete 16S rDNA sequences of 9 strains. Subsequent DNA/DNA reassociation studies and determination of the base composition of DNA identified seven isolates as Geobacillus thermodenitrificans, two isolates as G. thermoleovorans and one isolate as Bacillus pallidus. Four isolates represented two novel species of Bacillus. The remaining four represented novel Geobacillus species, one of which has recently been described as Bacillus vulcani DSMZ 13174 T.

A thermophilic Bacillus isolated from an Eolian shallow hydrothermal vent, able to produce exopolysaccharides.

A thermophilic aerobic microorganism, able to produce two exocellular polysaccharides (EPS1 and EPS2), was isolated from a shallow hydrothermal vent at Vulcano island (Eolian Islands, Italy). EPS1 and EPS2 were based on mannose and glucose although in a different ratio. EPS2 possessed a trisaccharide repeating unit with a manno-pyranoside configuration. New isolate phenotype was studied by physiological and morphological observations, including biochemical and antimicrobial susceptibility tests (134). Previous analyses carried out on 87 field isolates and 8 thermophilic reference bacilli displayed low phenotypic similarity level (S(SM) = 65%) with Bacillus thermodenitrificans DSM 465. Optimal growth occurs at 65 degrees C and pH 7.0. Oxidase and catalase are negative. The guanine-plus-cytosine (G+C) content of DNA is 52.7%. Genotypic investigations demonstrated the diversity of the isolate with fifteen selected thermophilic Bacillus spp. when we compared the restriction patterns of the amplified 16S rDNA. The membrane lipids are based on fatty acids mainly belonging to the iso-family.

Heterotrophic bacteria in the Ross Sea (Terra Nova Bay, Antarctica).

Microbial research on temporal variation of bacterial densities was carried out on seawater samples collected from two field stations at different depths during the Antarctic summer (oceanographic campaign 1989/1990). Bacterial densities evaluated on Marine Agar 2216 (Difco) and on TCBS Agar (Difco) after incubation at +4 degrees C for 21 days respectively ranged from 0 to 7.9 x 10(2) CFU/ml for heterotrophic bacteria and from 0 to 5.7 x 10(2) CFU/100ml for "presumptive vibrios". During the period of observation, Vibrio densities showed a higher variability than those of total heterotrophic bacteria. A high percentage of gelatinolytic and chitinolytic vibrios was observed. The qualitative composition of heterotrophic bacterial communities was studied on 38 morphological and biochemical characteristics of 152 strains isolated from the stations. The data were subsequently used to determine the structure and metabolic potentialities of bacterial communities in the two sites. Almost all the heterotrophic, psychrotrophic isolates were non fermentative Gram-negative rods, belonging to the genera Pseudomonas/Alcaligenes, Flavobacterium/Cytophaga. The bacterial communities in the two coastal habitats investigated were clearly different.

Psychrotrophic bacteria from a coastal station in the Ross sea (Terra Nova Bay, Antarctica).

Seawater samples were collected from a fixed, coastal station in the Terra Nova Bay at different depths during the Xth Oceanographic Cruise in the 1994-95 Antarctic summer. Picoplanktonic abundance, estimated by direct counts in epifluorescence microscopy, ranged from 2.2 x 10(7) to 1.6 x 10(8) cells.l-1. The heterotrophic bacterial densities, evaluated on Marine Agar 2216 (Difco) after incubation at +4 degrees C for 21 days, ranged from 2 x 10(3) to 4.5 x 10(6) CFU.l-1. The qualitative composition of the heterotrophic bacterial community was studied on 64 morphological and biochemical characters of the 125 strains isolated. Heterotrophic, psychrotrophic isolates were tentatively identified at genus level as Pseudomonas, Vibrio, Acinetobacter, and Flavobacterium/Cytophaga. In order to compare the characteristics of the isolates with those previously studied during 1989/90, the synthetical indices of the structure and the metabolic potentiality of the heterotrophic bacterial community were processed. Results showed that the bacterial community was metabolically more active and more homogenous than that previously studied.

Isolation of Arcobacter spp. from a brackish environment.

Arcobacter spp. were isolated from water and mussels of two brackish lakes near Messina (Italy). The isolates were phenotypically characterized on the basis of a large battery of cultural and biochemical tests. By comparison with the reference strains Arcobacter butzleri ATCC 49616 and A. cryaerophilus ATCC 43157 they may be considered Arcobacter butzleri-like bacteria. The current isolation suggests that the brackish environment may play an important role in the survival and transmission of Arcobacter spp. also by seafood cultured in the examined waters.

In vitro evaluation of antibacterial activity of Asparagopsis taxiformis from the Straits of Messina against pathogens relevant in aquaculture.

Ethanol extracts of Asparagopsis taxiformis collected from the Straits of Messina (Italy) were screened for antibacterial activity against pathogenic shellfish and fish bacteria previously isolated from local marine and brackish environments. Genetic labelling by DNA barcoding allowed us to identify the algal population as a biogeographical strain conspecific to A. taxiformis. The extract obtained in May showed the broadest antibacterial activity against all tested pathogenic bacteria, especially against Vibrio alginolyticus, Vibrio vulnificus and Aeromonas salmonicida subsp. salmonicida. Moderate activity was observed against Photobacterium damselae subsp. damselae and Photobacterium damselae subsp. piscicida, Salmonella sp., Vibrio cholerae, Vibrio harveyi and Vibrio parahaemolyticus. The absence of cytotoxic effects of active algal extracts was verified using trypan blue exclusion test on cells of digestive glands of Mytilus galloprovincialis. The results indicated that ethanol extracts of A. taxiformis could represent a source of antibacterial substances with potential use in aquaculture.

Occurrence of Helicobacter pylori DNA in the coastal environment of southern Italy (Straits of Messina).

AIMS: The occurrence of Helicobacter pylori in the coastal zone of the Straits of Messina (Italy) as free-living and associated with plankton was studied. METHODS AND RESULTS: Monthly sampling of seawater and plankton was carried out from April 2002 to March, 2003. All environmental samples analysed by cultural method, did not show the presence of H. pylori. The DNA extracted from all environmental samples was tested by PCR by using primers for H. pylori 16S rRNA, ureA and cagA. 16S rRNA PCR yielded amplified products of 522-bp in 15 of 36 (41.7%) of the environmental samples. By using the ureA primers to amplify the urea signal sequences, the predicted PCR products of 491-bp were obtained from eight (22.2%) of 36 environmental samples. PCR with cagA primers yielded amplified products of 349-bp in DNA extracted of seven of 36 (19.4%) of the environmental samples. When 16S rRNA, ureA and cagA amplified gene sequences were aligned with H. pylori 26695 and J99 genome sequences, we obtained a percentage of alignment over 90%. CONCLUSIONS: The detection of H. pylori genes in marine samples allows us to consider the marine environment a possible reservoir for this pathogenic bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: The direct detection of H. pylori genes may be relevant in order to consider the marine environment as significant reservoir for this bacterium.

Identification of thermophilic and marine bacilli from shallow thermal vents by restriction analysis of their amplified 16S rDNA.

AIMS: In order to identify 73 thermophilic isolates from shallow, marine thermal vents of Eolian Islands, we compared their restriction patterns of amplified 16S rDNA with those of nine well described Bacillus species and eight Eolian Bacillus strains. METHODS AND RESULTS: This study allowed to assign 57 (78%) isolates to different Bacillus species. Nineteen field strains were recognised as representatives of four described species, namely B. thermodenitrificans, "B. caldolyticus", B. vulcani and B. stearothermophilus. The profiles of 38 isolates matched instead, those of seven Eolian strains (B. thermodenitrificans strain A2, B. licheniformis strain B3-15, and five novel species, represented by Bacillus strain 1bw, Bacillus strain 4-1, Bacillus strain 5-2, Bacillus strain 10-1, Bacillus strain 1as). Among the 16 unidentified isolates, seven restriction patterns were recognised. CONCLUSIONS: This study showed that restriction analysis of amplified 16S rDNA is useful for a rapid and reliable identification of strains belonging to described species as well as for recognition of new species. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed a high taxonomic diversity among the thermophilic bacilli isolated from Eolian Islands and a distinct distribution of the species within the Eolian hydrothermal vent system.

Bacillus vulcani sp. nov., a novel thermophilic species isolated from a shallow marine hydrothermal vent.

A thermophilic spore-forming bacterium was isolated from sediment of a shallow hydrothermal vent at Vulcano Island (Italy). After phenotypic and molecular analyses, it was identified as a novel Bacillus species, for which the name Bacillus vulcani is proposed. The type strain is strain 3s-1T (= DSM 13174T = CIP 106305T).