Teresa L. Maugeri

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.

Heterotrophic bacteria community and pollution indicators of mussel--farm impact in the Gulf of Gaeta (Tyrrhenian Sea).

Field studies were carried out to determine and compare the impact of organic loads due to the biodeposition of a mussel farm on the water quality and sediment in a coastal area of the Tyrrhenian Sea (Western Mediterranean). A total of five environmental and five microbial parameters were examined from March, 1997 to February, 1998 on a monthly basis at three stations: the first was located under the mussel farm, the second located at about 40 m away from the mussel farm, while the third designed as a control was at about 1-km. No clear changes in the physical characteristics of the water masses were observed, comparing the three sampling sites and the water column generally showed homogeneous conditions (in terms of temperature and salinity). Changes in density of aerobic heterotrophic bacteria, Escherichia coli and Enterococci in the water column are apparently independent from changes in environmental parameters. At all stations a constant significant correlation between temperature and presumptive Vibrio parahaemolyticus was reported suggesting that this abiotic factor exerted a major control on this bacterial group and its distribution in the water column is not related to the biodeposition of the mussel farm. The major impact identified was on the sediment where variations in bacterial abundance was observed. In the Mussel station sediment enrichment of organic compounds, and the consequent modification of the characteristics of the benthic environment, determined an increase in aerobic heterotrophic bacteria, and particularly of vibrios density (on average about 60%), suggesting that these bacteria are good indicators of organic enrichment.

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-

Benthic microbial indicators of fish farm impact in a coastal area of the Tyrrhenian Sea

Abstract We studied the impact of organic loads due to the biodeposition of a fish farm in a non-impacted coastal area of the Tyrrhenian Sea (Western Mediterranean). Sediment chemistry and benthic microbial community were investigated from July 1997 to February 1998 on monthly basis at two stations: one was located under the fish farm, while the second was about 1 km away, and served as a reference site. The presence of the cage induced rapid changes in the benthic conditions: the sediments were rapidly (after 6 weeks) reduced. A significant accumulation of biopolymeric carbon was observed beneath the cage both 2 weeks after the initial cage deployment, on non-impacted sediments, and 5-7 months after, and appeared to be related to the fish farm production cycle. The density of microbial communities beneath the fish farm increased only during the first month of farming activity (July) and partially 7 months after cage disposal (i.e. in January), when there was a significant sediment organic enrichment. Additional evidence of rapid impact of the fish farm on the benthic bacterial communities is provided by the increase in the numbers of autofluorescent microbial cells. The photosynthetic eukaryotic cells displayed a highly reduced contribution to total autofluorescent microbial density, indicating that their decrease is related with biopolymeric carbon accumulation, and probably was due also to “shadow effect” induced by suspended material, coming from the fish farm, on the environment below. We propose here, to apply the ratio of culturable heterotrophic bacteria to microbial direct counts (CFU/MDC) to detect fish farm impact. In cage sediments, organic enrichment and the consequent modification of the characteristics of the benthic environment, determined an increase in aerobic heterotrophicbacteria and vibrio density indicating that they are efficient colonizers of organic-rich sediments. Densities of Escherichia coli and Enterococci were not significantly higher than in the reference site, and are likely of terrestrial origin. Thus they cannot be used as specific indicators of fish farm impact.

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.