Bacterial community diversity assessment in municipal solid waste compost amended soil using DGGE and ARISA fingerprinting methods

Bacterial community structure and diversity of Tunisian agricultural soil treated with different amounts of municipal solid waste compost (MSWC) and other fertilizers were studied using DGGE and ARISA fingerprinting methods. Sequence analysis of dominant DGGE bands revealed the presence of three major clusters, Cytophaga/Flexibacter/Bacteroides (CFB) group, Proteobacteria and Acidobacteria group. Using ARISA profiles, dominant populations were assigned to low and high GC Gram positive bacteria, Cyanobacteria, Spirochetes and Cytophagales. The two methods revealed the absence of significant bacterial community shifts related to the different MSWC applications. Moreover, indigenous bacterial population of the used loam-clayey soil was observed to limit proliferation and survival of Proteobacteria, initially dominant in MSWC and farmyard manure. Effectiveness of the two methods for soil bacterial community studying was shown. While DGGE was more accurate for bacterial identification, ARISA was more practical for handling and rapid estimation of dominant bacteria.     

Ribotyping of vibrio populations associated with cultured oysters (Ostrea edulis)

The intraspecific variability of Vibrio splendidus, V. harveyi and V. tubiashii recovered from oysters (Ostrea edulis) collected at the Mediterranean coast near Valencia, Spain, was analyzed by ribotyping. The two former species represented the most abundant ones, and the third one was the only species described as pathogenic for oysters. A total of 115 environmental strains were studied, 84 of V. splendidus, 23 of V. harveyi and 8 of V. tubiashii. Chromosomal DNA was digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Ribotyping among natural populations of the three species rendered 5 to 9 bands, and showed a high genetic diversity, with a ratio no. of strains/no. of ribotypes between 1.1 and 1.5. Cluster analysis of V. splendidus ribotypes suggests a seasonal pattern of incidence, with those ribotypes corresponding to winter and spring samples being maintained in the oysters over the year.     

Molecular analysis of the recently described lizardfish Saurida lessepsianus (Synodontidae) from the Red Sea and the Mediterranean,with remarks on its phylogeny and genetic bottleneck effect

The opening of the Suez Canal in 1869 led to a massive influx of Red Sea species that invaded the Mediterranean; this was termed ‘Lessepsian migration'. Among these species was a species of lizardfish, identified by some authors as Saurida undosquamis and by others as S. macrolepis. Recently, the Red Sea and the Mediterranean populations were described according to external characteristics as a unique taxon, Saurida lessepsianus. Our molecular study confirms this finding and determines that all previous records of S. undosquamis and S. macrolepis in the Red Sea and the Mediterranean are misidentifications of S. lessepsianus. The Mediterranean population of S. lessepsianus exhibits a lower genetic variability than that of the Red Sea population, suggesting a bottleneck effect.     

The Arabian scad Trachurus indicus, a new Indo-Pacific species in the Mediterranean Sea

The Arabian scad Trachurus indicus is recorded for the first time from the Mediterranean Sea (Iskenderun Bay, Turkey). The presence of this Indo-Pacific fish in the Mediterranean Sea is probably because of migration from the Red Sea via the Suez Canal.     

Characterization of a deep-sea microbial mat from an active cold seep at the Milano mud volcano in the Eastern Mediterranean Sea

A white, filamentous microbial mat at the Milano mud volcano in the Eastern Mediterranean Sea was sampled during the Medinaut cruise of the R/V Nadir in 1998. The composition of the mat community was characterized using a combination of phylogenetic and lipid biomarker methods. The mat sample was filtered through 0.2 and 5-microm filters to coarsely separate unicellular and filamentous bacteria. Analyses of 16S rRNA gene sequences amplified from the total community DNA from these fractions showed that similar archaeal populations were present in both fractions. However, the bacterial populations in the fractions differed from one another, and were more diverse than the archaeal ones. Lipid analysis showed that bacteria were the dominant members of the mat microbial community and the relatively low delta(13)C carbon isotope values of bulk bacterial lipids suggested the occurrence of methane- and sulfide-based chemo(auto)trophy. Consistent with this, the bacterial populations in the fractions were related to Alpha-, Gamma- and Epsilonproteobacteria, most of which were chemoautotrophic bacteria that utilize hydrogen sulfide (or reduced sulfur compounds) and/or methane. The most common archaeal 16S rRNA gene sequences were related to those of previously identified Archaea capable of anaerobic methane oxidation. Although the filamentous organisms observed in the mat were not conclusively identified, our results indicated that the Eastern Mediterranean deep-sea microbial mat community might be sustained on a combination of methane- and sulfide-driven chemotrophy.     

New insights into metabolic properties of marine bacteria encoding proteorhodopsins

Proteorhodopsin phototrophy was recently discovered in oceanic surface waters. In an effort to characterize uncultured proteorhodopsin-exploiting bacteria, large-insert bacterial artificial chromosome (BAC) libraries from the Mediterranean Sea and Red Sea were analyzed. Fifty-five BACs carried diverse proteorhodopsin genes, and we confirmed the function of five. We calculate that proteorhodopsin-exploiting bacteria account for 13% of microorganisms in the photic zone. We further show that some proteorhodopsin-containing bacteria possess a retinal biosynthetic pathway and a reverse sulfite reductase operon, employed by prokaryotes oxidizing sulfur compounds. Thus, these novel phototrophs are an unexpectedly large and metabolically diverse component of the marine microbial surface water.     

Evidence for colonization and destruction of hinge ligaments in cultured juvenile Pacific oysters (Crassostrea gigas) by cytophaga-like bacteria.

Several strains of cytophaga-like gliding bacteria (CLB) were isolated as numerically dominant or codominant components of bacterial populations associated with proteinaceous hinge ligaments of cultured juvenile Pacific oysters, Crassostrea gigas. These bacteria were morphologically similar to long, flexible bacilli occurring within degenerative lesions in oyster hinge ligaments. Among bacteria isolated from hinge ligaments, only CLB strains were capable of sustained growth with hinge ligament matrix as the sole source of organic carbon and nitrogen. In vitro incubation of cuboidal portions of ligament resilium with ligament CLB resulted in bacterial proliferation on the surfaces and penetration deep into ligament matrices. Bacterial proliferation was accompanied by loss of resilium structural and mechanical integrity, including complete liquefaction, at incubation temperatures between 10 and 20 degrees C. The morphological, distributional, and degradative characteristics of CLB isolated from oyster hinge ligaments provide compelling, albeit indirect, evidence that CLB are the agents of a degenerative disease affecting juvenile cultured oysters. The motility, metabolic, and hydrolytic characteristics of hinge ligament CLB and the low moles percent G + C values (32.4 to 32.9) determined for three representative strains indicate that they are marine Cytophaga spp.     

Evidence for colonization and destruction of hinge ligaments in cultured juvenile Pacific oysters (Crassostrea gigas) by cytophaga-like bacteria

Several strains of cytophaga-like gliding bacteria (CLB) were isolated as numerically dominant or codominant components of bacterial populations associated with proteinaceous hinge ligaments of cultured juvenile Pacific oysters, Crassostrea gigas. These bacteria were morphologically similar to long, flexible bacilli occurring within degenerative lesions in oyster hinge ligaments. Among bacteria isolated from hinge ligaments, only CLB strains were capable of sustained growth with hinge ligament matrix as the sole source of organic carbon and nitrogen. In vitro incubation of cuboidal portions of ligament resilium with ligament CLB resulted in bacterial proliferation on the surfaces and penetration deep into ligament matrices. Bacterial proliferation was accompanied by loss of resilium structural and mechanical integrity, including complete liquefaction, at incubation temperatures between 10 and 20 degrees C. The morphological, distributional, and degradative characteristics of CLB isolated from oyster hinge ligaments provide compelling, albeit indirect, evidence that CLB are the agents of a degenerative disease affecting juvenile cultured oysters. The motility, metabolic, and hydrolytic characteristics of hinge ligament CLB and the low moles percent G + C values (32.4 to 32.9) determined for three representative strains indicate that they are marine Cytophaga spp.     

First record of the bluespotted cornetfish from the Mediterranean Sea

Three specimens of the Indo-Pacific Bluespotted cornetfish Fistularia commersonii are recorded for the first time from the Mediterranean. The presence of this species in the Mediterranean is due to migration from the Red Sea via the Suez Canal.     

Factors influencing in vitro killing of bacteria by hemocytes of the eastern oyster (Crassostrea virginica).

A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer. Vibrio vulnificus strains, opaque and translucent (with and without capsules), were examined only in summer. Among V. parahaemolyticus and L. monocytogenes, significantly (P < 0.05) higher levels of killing by hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P > 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster's cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.     

Apogon smithi (Kotthaus, 1970) (Teleostei: Apogonidae), a Red Sea cardinalfish colonizing the Mediterranean Sea

Smith's cardinalfish Apogon smithi is recorded for the first time in the Mediterranean Sea. Six specimens were caught by commercial trawler north of Jaffa, Israel, at depths of 40 m and the relatively high number of specimens suggests that A. smithi has established a sustainable population. This is the third Indo-Pacific apogonid species documented in the Mediterranean Sea, and is assumed to be a migrant originating from the Red Sea.     

Effect of dietary supplementation with a Saccharomyces cerevisiae mannan oligosaccharide on the bacterial community structure of broiler cecal contents

This study investigated the effects of dietary supplementation with a prebiotic mannan oligosaccharide (MOS) on broiler performance, bacterial community structure, and phylogenetic populations of cecal contents. Bird performance data were collected, and cecal samples were extracted from randomly caught poults from each treatment group every 7 days from hatching to the age of 42 days. Weight gain, feed consumption, and feed efficiency ratios did not differ significantly between groups. Automated ribosomal intergenic spacer analysis (ARISA) of the bacterial communities in birds receiving MOS-supplemented diets indicated that dietary supplementation with MOS at either of 2 levels significantly altered the bacterial community structure from that of the control group on all sample days. The phylogenetic identities of bacteria contained within the cecum were determined by constructing a 16S rRNA gene clone library. A total of 594 partial 16S rRNA gene sequences from the cecal contents were analyzed and compared for the three dietary treatments. The dominant bacteria of the cecum belonged to three phyla, Firmicutes, Bacteroidetes, and Proteobacteria; of these, Firmicutes were the most dominant in all treatment groups. Statistical analysis of the bacterial 16S rRNA gene clone libraries showed that the compositions of the clone libraries from broilers receiving MOS-supplemented diets were, in most cases, significantly different from that of the control group. It can be concluded that in this trial MOS supplementation significantly altered the cecal bacterial community structure.     

rDNA analysis of the Red Sea seagrass,Halophila, reveals vicariant evolutionary diversification

The effects of opening the Suez Canal as a connection between the Red Sea and the Mediterranean Sea were reported for a number of marine species. However, the evolutionary origin of the seagrasses in the Red Sea and the linking population genetics of seagrasses between the Arabian Sea, the Gulf of Aden, the Red Sea and the Mediterranean Sea have not yet been investigated in detail. The invasion of Halophila stipulacea Asch. from the Red Sea into the Mediterranean Sea after the opening of the Suez Canal was already recorded. We hypothesize that Halophila ovalis populations in the Red Sea developed through long-term historical processes such as vicariant evolutionary diversification. Seagrass samples were collected along the Egyptian coastline of the Red Sea and analysed by the molecular marker ITS. The sequences were compared with published ITS sequences from seagrasses collected in the whole area of interest. In this study, we reveal the linking population genetics, phylogeography and phylogenetics of two dominant seagrass species, Halophila stipulacea and Halophila ovalis, among species collected in the Red Sea and worldwide. The results indicate that the Red Sea Halophila ovalis populations do not group to Halophila ovalis worldwide, and Halophila major, Halophila ovalis collected worldwide and Halophila ovalis collected at the Red Sea are sister clades. Hence, vicariant evolutionary diversification for Halophila ovalis may occur in the Red Sea.     

Aerobic and facultative anaerobic heterotrophic bacteria associated to Mediterranean oysters and seawater.

A comparative study on the composition and seasonal fluctuations of the main heterotrophic bacterial groups and species isolated from Mediterranean oysters and their growing-seawater was carried out. For the study we used 574 strains isolated from Marine Agar (MA) and submitted to numerical analysis of phenotypic traits in previous studies, plus 323 isolates recovered on Thiosulphate Citrate Bile Sucrose (TCBS) agar from the same samples and identified in this study. Oyster samples were dominated by halophilic fermentative bacteria during most of the year with predominance of two Vibrio species, V. splendidus (at temperatures lower than 20 degrees C), and V. harveyi (at higher temperatures). On the contrary, Vibrio spp. was not the predominant microbiota of seawater, where most isolates had remained unidentified but corresponded to alpha-Proteobacteria, as shown by rDNA hybridization with phylogenetic probes in this study. Among the strict aerobes that could be identified, none of them showed a clear dominance, and many different groups were represented in very low percentages, in contrast with the major species from oyster samples. Shannon-Weaver diversity index revealed significant differences between both types of samples. No apparent seasonality was found in the distribution of seawater species, in sharp contrast with oyster-associated bacteria.     

A Vibrio splendidus strain is associated with summer mortality of juvenile oysters Crassostrea gigas in the Bay of Morlaix (North Brittany, France).

Juvenile oysters Crassostrea gigas cultured in the Bay of Morlaix (France) have suffered unexplained summer mortalities for over a decade. In the present study, we tested the hypothesis that a bacterial pathogen could be responsible for this phenomenon. A first attempt failed to isolate a bacterial pathogen from moribund or weak oysters. Only non-pathogenic, probably opportunistic, bacteria were isolated. As an alternative approach, we focused on oysters presenting reduced stress-response capacities (determined by circulating noradrenaline measurements), a characteristic of juvenile oysters entering an early phase of the disease. Cultures of bacterial isolates on TCBS plates revealed that a Vibrio strain was present in diseased oysters and scarce or absent in healthy oysters. Experimental infections indicated that this Vibrio can cause mortalities of juvenile oysters when injected at concentrations ranging from 10(4) to 10(8) CFU oyster(-1). Similarly to the summer mortality disease, the Vibrio isolate caused higher mortalities at higher temperatures; apparently, it could not be transmitted horizontally, it did not affect adult oysters and it induced stress-response dysfunctions in juvenile oysters. Phenotypic and genotypic characterizations identified the pathogen as Vibrio splendidus. Taken together, the present results satisfy Koch's postulate and suggest that this bacterial strain is probably responsible for the juvenile oyster summer mortalities in the Bay of Morlaix.     

Bacteria in bivalve shellfish with special reference to the oyster

K ueh , C.S.W. & C han , K-Y. 1985. Bacteria in bivalve shellfish with special reference to the oyster. Journal of Applied Bacteriology , 59 , 41–47.
The bacterial flora of the Pacific oyster Crassostrea gigas , the sea mussel Perna viridis and the arkshell clam Scapharca cornea differed considerably from that of seawater in both numbers and generic composition. The numbers of heterotrophic bacteria in the bivalve shellfish, including the anaerobes and spore-forming bacteria, were greater than that in the surrounding water. Pseudomonas spp. were the dominant organisms, comprising over one third of the 321 strains characterized after isolation from the bivalves and seawater. Other bacteria isolated from the shellfish included Vibrio, Acinetobacter , and Aeromonas spp., whereas the seawater flora consisted mainly of coliform organisms, coryneform bacteria and Flavobacterium/ Cytophaga spp. Bacteria associated with the deposit-feeding clams were higher in density and more distinct in generic composition as compared with those in the suspension-feeding oysters and mussels. Over 90% of the coliform and heterotrophic bacteria in oysters were found in organs associated with the digestive tract. Coliforms were mainly found in the stomach while heterotrophs were present in both stomach and the lower intestine. The results suggest that the stomach flora of oysters are mainly derived from the external environment and, through a process of selection and multiplication, that it may be gradually replaced by a more indigenous population which dominates the lower digestive tract.     

Bacterial 16S rRNA Gene Analysis Revealed That Bacteria Related to Arcobacter spp. Constitute an Abundant and Common Component of the Oyster Microbiota (Tiostrea chilensis)

To explore the bacterial microbiota in Chilean oyster (Tiostrea chilensis), a molecular approach that permits detection of different bacteria, independently of their capacity to grow in culture media, was used. Bacterial diversity was assessed by analysis of both the 16S rDNA and the 16S-23S intergenic region, obtained by PCR amplifications of DNA extracted from depurated oysters. RFLP of the PCR amplified 16S rDNA showed a prevailing pattern in most of the individuals analyzed, indicating that a few bacterial species were relatively abundant and common in oysters. Cloning and sequencing of the 16S rDNA with the prevailing RFLP pattern indicated that this rRNA was most closely related to Arcobacter spp. However, analysis by the size of the amplified 16S-23S rRNA intergenic regions revealed not Arcobacter spp. but Staphylococcus spp. related bacteria as a major and common component in oyster. These different results may be caused by the absence of target for one of the primers employed for amplification of the intergenic region. Neither of the two bacteria species found in large abundance was recovered after culturing under aerobic, anaerobic, or microaerophilic conditions. This result, however, is expected because the number of bacteria recovered after cultivation was less than 0.01% of the total. All together, these observations suggest that Arcobacter-related strains are probably abundant and common in the Chilean oyster bacterial microbiota.     

Uptake and clearance of Vibrio vulnificus from Gulf coast oysters (Crassostrea virginica)

Oysters collected in late winter, when they were free of Vibrio vulnificus, were exposed in the organism in the laboratory. The oysters effectively concentrated the bacteria from seawater, but when the inoculum was removed, the bacteria were rapidly cleared from the oyster tissues. These results suggest that V. vulnificus may be found in oysters as a result of filtration of the bacteria from seawater rather than active multiplication of the bacteria in the oysters.     

Uptake and clearance of Vibrio vulnificus from Gulf coast oysters (Crassostrea virginica).

Oysters collected in late winter, when they were free of Vibrio vulnificus, were exposed in the organism in the laboratory. The oysters effectively concentrated the bacteria from seawater, but when the inoculum was removed, the bacteria were rapidly cleared from the oyster tissues. These results suggest that V. vulnificus may be found in oysters as a result of filtration of the bacteria from seawater rather than active multiplication of the bacteria in the oysters.