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The bacterial community structure of activated sludge of a large municipal wastewater treatment plant was investigated by use of the rRNA approach. Almost-full-length genes coding for the small-subunit rRNA (rDNA) were amplified by PCR and subsequently cloned into the pGEM-T vector. Clones were screened by dot blot hybridization with group-specific oligonucleotide probes. The phylogenetic affiliations of clones were compared with the results obtained with the original sample by in situ hybridization with fluorescently labeled, rRNAtargeted oligonucleotide probes and found to be in general agreement. Twenty-five 16S rDNA clones were fully sequenced, 11 were almost fully (>80%) sequenced, and 27 were partially sequenced. By comparative sequence analyses, the majority of the examined clones (35 of 67) could be affiliated with the beta subclass of the class Proteobacteria. The gamma and alpha subclasses of Proteobacteria were represented by 13 and 4 clones, respectively. Eight clones grouped with the epsilon group of Proteobacteria, and five clones grouped with gram-positive bacteria with a low DNA G؉C content. The 16S rDNA of two clones showed similarity with 16S rDNA genes of members of the phyla Chlamydiae and Planctomyces. 16S rRNA-targeted oligonucleotide probes were designed and used for the enumeration of the respective bacteria. Interestingly, potentially pathogenic representatives of the genus Arcobacter were present in significant numbers (4%) in the activated sludge sample examined. Pairs of probes targeted to the 5 and 3 regions were used for detection of chimeric sequences by in situ hybridization. Two clones could be identified as chimera by applying such a pair of probes.

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Viral vectors based on various naturally occurring adeno-associated virus (AAV) serotypes are among the most promising tools in human gene therapy. For the production of recombinant AAV (rAAV) vectors, researchers are focusing predominantly on cross-packaging an artificial AAV genome based on serotype 2 (AAV2) into capsids derived from other serotypes. Within the packaged genome the inverted terminal repeats (ITRs) are the only cis-acting viral elements required for rAAV vector generation and depict the lowest common denominator of all AAV2-derived vector genomes. Up to now, no quantitative PCR (qPCR) for the detection and quantification of AAV2 ITRs could be established because of their extensive secondary hairpin structure formation. Current qPCR-based methods are therefore targeting vector-encoded transgenes or regulatory elements. Herein we establish a molecular biological method that allows accurate and reproducible quantification of AAV2 genomes on the basis of an AAV2 ITR sequence-specific qPCR. Primers and labeled probe are located within the ITR sequence and have been designed to detect both wild-type AAV2 and AAV2-based vectors. This method is suitable for detecting single-stranded DNA derived from AAV2 vector particles and double-stranded DNA derived from vector plasmids. The limit of detection has been determined as 50 ITR sequence copies per reaction, by comparison with a plasmid standard. In conclusion, this method describes the first qPCR system facilitating the detection and quantification of AAV2 ITR sequences. Because this method can be used universally for all AAV2 genome-based vectors, it will significantly simplify rAAV2 vector titrations in the future.

Aims: To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine. Methods and Results: Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4 ¢ ,6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were £2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50-90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were £2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium. Conclusions: Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, microorganisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish. Significance and Impact of the Study: Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.

To study the possible use of probiotics in fish farming, we evaluated the in vitro and in vivo antagonism of antibacterial strainPseudomonas fluorescens strain AH2 against the fish-pathogenic bacterium Vibrio anguillarum. As iron is important in virulence and bacterial interactions, the effect ofP. fluorescens AH2 was studied under iron-rich and iron-limited conditions. Sterile-filtered culture supernatants from iron-limited P. fluorescens AH2 inhibited the growth ofV. anguillarum, whereas sterile-filtered supernatants from iron-replete cultures of P. fluorescens AH2 did not.P. fluorescens AH2 inhibited the growth of V. anguillarum during coculture, independently of the iron concentration, when the initial count of the antagonist was 100 to 1,000 times greater that of the fish pathogen. These in vitro results were successfully repeated in vivo. A probiotic effect in vivo was tested by exposing rainbow trout (Oncorynchus mykissWalbaum) to P. fluorescens AH2 at a density of 105 CFU/ml for 5 days before a challenge with V. anguillarum at 104 to 105 CFU/ml for 1 h. Some fish were also exposed to P. fluorescens AH2 at 107 CFU/ml during the 1-h infection. The combined probiotic treatment resulted in a 46% reduction of calculated accumulated mortality; accumulated mortality was 25% after 7 days at 12°C in the probiotic-treated fish, whereas mortality was 47% in fish not treated with the probiont.

Enterococci have gained significance as the cause of nosocomial infections; they occur as food contaminants and have also been linked to dental diseases. E. faecalis has a great potential to spread virulence as well as antibiotic resistance genes via horizontal gene transfer. The integration of food-borne enterococci into the oral biofilm in-vivo has been observed. Therefore, we investigated the virulence determinants and antibiotic resistance of 97 E. faecalis isolates from the oral cavity, food, and clinical specimens. In addition, phenotypic expression of gelatinase and cytolysin were tested, in-vitro biofilm formation was quantified and isolates were compared for strain relatedness via pulsed field gel electrophoresis (PFGE). Each isolate was found to possess two or more virulence genes, most frequently gelE, efaA, and asa1. Notably, plaque/saliva isolates possessed the highest abundance of virulence genes, the highest levels of phenotypic gelatinase and hemolysin activity and concurrently a high ability to form biofilm. The presence of asa1 was associated with biofilm formation. The biofilm formation capacity of clinical and plaque/saliva isolates was considerably higher than that of food isolates and they also showed similar antibiotic resistance patterns. These results indicate that the oral cavity can constitute a reservoir for virulent E. faecalis strains possessing antibiotic resistance traits and at the same time distinct biofilm formation capabilities facilitating exchange of genetic material.

We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment surface at 110 m by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes of various specificity. In addition, we experimentally examined the turnover of dissolved amino acids and carbohydrates together with the microbial colonization of aggregates formed in rolling tanks in the lab. Generally, between 40 and more than 80% of the microbes enumerated by DAPI staining (4,6-diamidino-2-phenylindole) were detected as Bacteria by the probe EUB338. At a depth of 25 m, 10.5% ؎ 7.9% and 14.2% ؎ 10.2% of the DAPI cell counts were detected by probes specific for ␣-and ␤-Proteobacteria. These proportions increased to 12.0% ؎ 3.3% and 54.0% ؎ 5.9% at a depth of 50 m but decreased again at the sediment surface at 110 m to 2.7% ؎ 1.4% and 41.1% ؎ 8.4%, indicating a clear dominance of ␤-Proteobacteria at depths of 50 and 110 m, where aggregates have an age of 3 to 5 and 8 to 11 days, respectively. From 50 m to the sediment surface, cells detected by a Cytophaga/Flavobacteria-specific probe (CF319a) comprised increasing proportions up to 18% of the DAPI cell counts. ␥-Proteobacteria always comprised minor proportions of the aggregate-associated bacterial community. Using only two probes highly specific for clusters of bacteria closely related to Sphingomonas species and Brevundimonas diminuta, we identified between 16 and 60% of the ␣-Proteobacteria. In addition, with three probes highly specific for close relatives of the ␤-Proteobacteria Duganella zoogloeoides (formerly Zoogloea ramigera), Acidovorax facilis, and Hydrogenophaga palleroni, bacteria common in activated sludge, 42 to 70% of the ␤-Proteobacteria were identified. In the early phase (<20 h) of 11 of the 15 experimental incubations of aggregates, dissolved amino acids were consumed by the aggregate-associated bacteria from the surrounding water. This stage was followed by a period of 1 to 3 days during which dissolved amino acids were released into the surrounding water, paralleled by an increasing dominance of ␤-Proteobacteria. Hence, our results show that lake snow aggregates are inhabited by a community dominated by a limited number of ␣-and ␤-Proteobacteria, which undergo a distinct succession. They successively decompose the amino acids bound in the aggregates and release substantial amounts into the surrounding water during aging and sinking.