Concetta Gugliandolo

Distribution of potentially pathogenic bacteria as free living and plankton associated in a marine coastal zone

Aims:  To determine the abundance of faecal and nonfaecal bacteria related to human and animal health, as free living or associated with small (>64 μm) and large (>200 μm) plankton, samples were collected monthly from the coastal zone at Messina (Italy).

Detection of Arcobacter spp. in the Coastal Environment of the Mediterranean Sea

ABSTRACT The occurrence of Arcobacter spp. was studied in seawater and plankton samples collected from the Straits of Messina, Italy, during an annual period of observation by using cultural and molecular techniques. A PCR assay with three pairs of primers targeting the 16S and 23S rRNA genes was used for detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii in cultures and environmental samples. Only one of the Arcobacter species, A. butzleri, was isolated from seawater and plankton samples. With some samples the A. butzleri PCR assay gave amplified products when cultures were negative. A. cryaerophilus and A. skirrowii were never detected by culture on selective agar plates; they were detected only by PCR performed directly with environmental samples. Collectively, our data suggest that culturable and nonculturable forms of Arcobacter are present in marine environments. The assay was useful for detecting Arcobacter spp. both as free forms and intimately associated with plankton. This is the first report showing both direct isolation of A. butzleri and the presence of nonculturable Arcobacter spp. in the coastal environment of the Mediterranean Sea.

Potentially pathogenic vibrios in brackish waters and mussels.

Water and mussel samples were collected from two brackish lakes, used as mussel farms, at different times of the year, for the quantitative analysis of Vibrio spp. and for the isolation of potentially pathogenic species. The isolates underwent cultural and biochemical tests selected for rapid identification. Glucose oxidizing‐fermenting and O/129 sensitive strains were distinguished on the basis of the following tests: sucrose and cellobiose utilization, sulphatase activity and polymyxin B resistance performed, respectively, on TCBS, CPC and SPS media. Responses to the presence of β‐galactosidase, salt requirement and growth on triple sugar iron medium were also added. A total of 125 from 152 isolates were referred to the species Vibrio fluvialis (55 strains), V. alginolyticus (40), V. parahaemolyticus (11), V. vulnificus (10) and V. mimicus (9). The remaining 27 isolates were not identified. The isolation of potentially pathogenic vibrios from cultivated mussels is a risk for health of people consuming raw seafood. Therefore, a long‐term monitoring programme should also include the search for these bacterial species.

An exopolysaccharide produced by Geobacillus thermodenitrificans strain B3-72: Antiviral activity on immunocompetent cells

The immunomodulatory and antiviral effects of an extracellular polysaccharide (EPS-2), produced by a strain of Geobacillus thermodenitrificans isolated from a shallow marine vent of Vulcano Island (Italy), were evaluated. In the present study, we show for the first time that EPS-2 treatment hinder HSV-2 replication in human peripheral blood mononuclear cells (PBMC) but not in WISH cells. In fact, high levels of IFN-alpha, IL-12, IFN-gamma, TNF-alpha, IL-18 were detected in supernatants of EPS-2 treated PBMC. Moreover, this effect was dose-dependent. Taken together, our results confirm that the immunological disorders determined by HSV-2 could be partially restored by treatment with EPS-2.

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.

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).

Pathogenic Vibrio, Aeromonas and Arcobacter spp. associated with copepods in the Straits of Messina (Italy).

In aquatic environments bacteria have a strong affinity for a variety of substrates including animal surfaces. The chitinous skeletons of crustaceans are nutrient sources that encourage bacterial attachment and colonization in marine environment (Sieburth, 1975). Bacteria associated with plankton can survive in adverse environmental conditions longer than free-living forms (Carman and Dobbs, 1997). Zooplankton carry a natural commensal microflora attached to external surfaces and in the digestive tract, mainly composed of Vibrio spp. (Huq et al., 1983; Tamplin et al., 1990). The interest in the occurrence and distribution of vibrios as free living and zooplankton associated involves ecological and epidemiological aspects. The genus Vibrio currently includes 72 species, among these species Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus account for the majority of Vibrio infections in humans (Farmer et al., 2003). The association of potentially pathogenic vibrios with marine zooplankton has been demonstrated in the Mediterranean Sea for Vibrio alginolyticus, V. cholerae nonO1, V. fluvialis, V. parahaemolyticus and V. vulnificus in different temperature and salinity conditions (Carli et al., 1993; Pruzzo et al., 1996; Montanari et al., 1999; Maugeri et al., 2000a, 2004, 2006; Gugliandolo et al., 2005). While the colonization of copepods, which represent the main component of zooplankton, by vibrios, is a well described phenomenon, little is known for other potentially pathogenic bacteria for humans and animals. Aeromonas spp. are ubiquitous inhabitants of aquatic ecosystems such as freshwater, coastal water and sewage (Monfort and Baleux, 1990; Krovacek et al., 1994). Members of the Aeromonas genus are recognized as pathogens for humans (Janda and Abbott, 1998) and animals including amphibians, reptiles, and fish (Austin and Austin, 1997). Strains of Aeromonas hydrophila, Aeromonas sobria, Aeromonas caviae, Aeromonas veronii and Aeromonas salmonicida have been isolated from coastal waters in Italy (Fiorentini et al., 1998; Sechi et al., 2002). Few reports have dealt with aeromonads associated with marine zooplankton (Alfredsson et al., 1995; Montanari et al., 1999; Dumontet et al., 2000). Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii, included in the family of Campylobacteraceae, have been associated with human and animal enteric diseases (Vandamme et al., 1992) and they are con-

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.

Three novel halotolerant and thermophilic Geobacillus strains from shallow marine vents.

During a polyphasic taxonomic analysis performed on isolates from shallow marine hydrothermal vents of Eolian Islands (Italy), three thermophilic, halotolerant bacilli, designated as strain 1bw, strain 5-2 and strain 10-1, could not be affiliated to any described species. Physiological and biochemical characteristics, membrane lipids composition, mol % G+C content, and phylogenetic relationships determined on the basis of the 16S rRNA gene sequence analysis, placed these strains within the genus Geobacillus. The three strains were only moderately related to species of Geobacillus and their relatives, members of Saccharococcus. Determination of the relatedness among each other at a higher taxonomic level by DNA-DNA reassociation experiments demonstrated the three isolates to represent three different novel Geobacillus genomospecies. The taxonomic novelty of these three marine strains was substantiated by their physiological properties and by fatty acid patterns that did not match closely those of any Geobacillus type strain. These three novel strains could be of interest to biotechnology because of their ability to produce exopolysaccharides and to adhere on polystirene, characteristics undescribed so far for other Geobacillus species. They are also able to utilise hydrocarbons such as gas oil, kerosene and mineral lubricating oil. Strain 5-2 is tolerant to zinc.

Bacillus aeolius sp. nov. a Novel Thermophilic, Halophilic Marine Bacillus Species from Eolian Islands (Italy)

Phylogenetic relationships of a thermophilic, halophilic, aerobic spore-forming strain 4-1(T), isolated from the water of a shallow sea hot spring at Vulcano Island (Italy), revealed its relatedness to members of the genus Bacillus. Chemotaxonomic and phenotypic properties of strain 4-1(T) are sufficiently different from related moderately thermophilic species, e.g., B. smithii, B. fumarioli, B. oleronius, B. sporothermodurans and B. infernus to describe strain 4-1(T) as a new Bacillus species, for which the name Bacillus aeolius sp. nov. is proposed. Strain 4-1(T) is characterised by the potential biotechnological important properties such as exopolysaccharide production, surfactant activity, and utilisation of hydrocarbons.