S. Botella

Specific Detection of Arcobacter and Campylobacter Strains in Water and Sewage by PCR and Fluorescent In Situ Hybridization

ABSTRACT The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting Arcobacter and Campylobacter strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 103 cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples.

Direct Detection and Identification of Arcobacter Species by Multiplex PCR in Chicken and Wastewater Samples from Spain

Twenty-two chicken livers, 10 chicken carcasses, and 15 wastewater samples were processed and analyzed for Arcobacter by PCR and traditional culture methods. Samples were enriched for 24 and 48 h, incubated at 30 degrees C under aerobic conditions, and streaked on blood selective media. To determine the best isolation conditions, 20 samples also were processed under microaerophilic conditions at 37 degrees C. Simple and multiplex PCR assays were used directly with enrichment broths and isolated strains. Seventeen Arcobacter strains were isolated from chicken samples, and A. butzleri was the only Arcobacter species identified. The direct PCR assay revealed that 29 of the 32 chicken samples were contaminated with Arcobacter. A. butzleri was the most frequently detected species, although Arcobacter cryaerophilus also was present in some of the samples and Arcobacter skirrowii occasionally was detected. All the wastewater samples were positive by PCR assay for Arcobacter after 24 h of enrichment. A. butzleri and A. cryaerophilus were detected with the multiplex PCR assay. Fourteen Arcobacter strains were isolated from 10 of the 15 water samples analyzed; 7 were identified as A. butzleri and the remaining 7 were A. cryaerophilus. Both for chicken and water samples, Arcobacter detection rate for PCR amplification was higher than for culture isolation. These results indicate the high prevalence of Arcobacter in chicken and wastewater and the inadequacy of available cultural methods for its detection. The species-specific multiplex PCR assay is a rapid method for assessing Arcobacter contamination in chicken and wastewater samples and is a viable alternative to biochemical identification of isolated strains.

Amplified fragment length polymorphism (AFLP) and biochemical typing of Photobacterium damselae subsp. damselae

Aims: The aim of the present study was to characterize subspecifically Photobacterium damselae subsp. damselae strains isolated from cultured Sparus aurata and Dicentrarchus labrax by means of phenotypic and molecular typing techniques (amplified fragment length polymorphism, AFLP). 
Methods and Results: Seventy‐one strains of P. damselae subsp. damselae were isolated from 38 cultured fishes at different fish farms located on the Mediterranean coast near Valencia, Spain. Most fish studied were asymptomatic and some were recovered during infectious outbreaks. Phenotypic characterization revealed a considerable degree of variability within the subspecies, including some characters, such as production of urease, which are used to differentiate P. damselae subsp. damselae from P. damselae subsp. piscicida. Genetic characterization was conducted on a selection of 33 strains, including two reference strains. Dice coefficient (Sd) and the unweighted pair group method with average linkage (UPGMA) were used for numerical analysis of banding patterns. AFLP type was defined on the basis of 100% similarity in the dendrogram obtained, yielding 24 distinct AFLP profiles. At 70% similarity, 13 clusters were defined, thus confirming the great variability observed for the phenotypic traits. 
Conclusions: The AFLP variability shown by the isolates was high enough to discriminate between different strains which colonize the same fish. However, closely related AFLP types were usually derived from strains isolated at the same fish farm, indicating an epidemiological relationship. 
Significance and Impact of the Study: This study has confirmed that the AFLP technique allows discrimination of individual strains within P. damselae subsp. damselae for epidemiological studies, and that this subspecies exhibits greater variability than that described for subspecies piscicida.

Molinate decontamination processes in effluent water from rice fields

The performance of aeration, photodecomposition and biological degradation processes as methods to reduce molinate contamination levels in effluent water from rice fields was studied. Aeration produced a molinate dissipation of 84%, as against 22% without aeration. Application of UV-light to clean water solutions achieved a molinate photodecomposition of 96% in 24 h. Maximal degradation obtained in algal cultures was 55% in 20 days and 78% in 40 days. In micro-organism cultures, kept in darkness and with a continuous flow of aqueous solution of molinate and inorganic salts, a degradation of 97% was achieved.

Identification of actinomycetes with antifungal activity isolated from soil amended with composts

The search for new antifungal biocontrol strategies to inhibit the growth of phytopathogenic fungi has been focused on the genus Streptomyces and related species. Actinomycetes were isolated on starch casein agar (SCA), arginine glycerol salts agar (AGSA), and glycerol asparagine agar (GAA), and co-cultured with phytopathogenic fungi in potato dextrose agar (PDA) and yeast extract malt extract agar (YMA). In this work, we have isolated 52 actinomycete strains, 13 of them showing high inhibitory activity against the phytopathogenic fungi tested. Isolated strains were identified by chemotaxonomic procedures, 16S rDNA sequences analysis, and morphological methods. These strains belongs to the species Streptomyces variegatus, S. griseoruber, S. lusitanus, S. roseogriseus, S. coeruleorubidus, S, griseoruber, S. lincolensis, S. aureoverticilatus, S. speibonae, and Lechevalieria xinjiangensis.