Azevedo, N. F.

Gião, M. S.

Keevil, C. W.

Vieira, M. J.

Wilks, S. A.

Universidade do Minho: RepositoriUM

Survival of VBNC legionella pneumophila in drinking water following chlorine disinfection

163P O S T E R S6. Environmental Microbioloy162P O S T E R S6. Environmental MicrobioloyP.ENV.53 Microbial communities in the cooling water system of a SpanishNuclear Power PlantGarcía AM1, Sarró MI1, Fernández MA2, Moreno DA11Dep. Ingeniería y Ciencia de los Materiales, 2Dep. Ingeniería Química Industrial y del Medio Ambiente, ETSIIndustriales, Universidad Politécnica de Madrid, José GutiérrezAbascal 2,E-28006, Madrid, SpainIn the present research, the microbial communities from the coolingwater system of a Spanish Nuclear Power Plant have been studied toanalyse the system’s influence on the natural water used inrefrigeration. Traditional culture and molecular microbiologytechniques such as PCR, DGGE and DNA sequencing, as well as lightand epifluorescence microscopy have been used.The number of microorganisms found in the cooling water systemagrees with that detected in freshwater by other authors. No differencesare found throughout the cooling water system. However, variations inthe composition of the microbial population have been observeddepending on the season and at which end of the cooling water systemthe test was carried out. Among the microorganisms that wereidentified, some belonged to the phylogenetic groups of α - β - and δ -Proteobacteria, Bacillus-Staphylococcus, Actinomycetales,Deinococcus-Thermus, Bacteroidetes and Clostridiales. The results show the bacterial diversity found in these kinds ofindustrial water systems and the importance of the moleculartechniques used to study microbial communities.P.ENV.54 Survival of VBNC Legionella pneumophila in drinking waterfollowing chlorine disinfectionGião MS1,2, Azevedo NF1,2, Wilks SA2, Vieira MJ1, Keevil CW21CEB, Universidade do Minho, Braga, Portugal and 2SOBS, University ofSouthampton, Southampton, UK (salome.giao@deb.uminho.pt)Legionella pneumophila is a waterborne pathogen that has been foundto be resistant to the chlorine disinfection of drinking water. The aim ofthis work is to demonstrate that although no L. pneumophila can bedetected by using standard plating techniques, VBNC cells can stillsurvive in water systems. A cell suspension of L. pneumophila NCTC 12821 was prepared indechlorinated and filter-sterilised drinking water. Chlorine was addedand samples were taken after 0, 10, 20 and 30 minutes for cellquantification by culture on BCYE agar plates and by SYTO 9/PIdouble staining. In the control assay (with no chlorine) the number of cultivable andviable cells remained constant with time. When exposed to 0.2 mg L-1 of chlorine it was observed that cell cultivability decreased with time,but the number of viable cells remained constant. When chlorineconcentration was increased to 0.7 mg L-1 and 1.2 mg L-1,cultivability was completely lost in 30 and 10 minutes, respectively. Inboth cases there was no significant decrease in the number of viablecells and no DNA cuts were detected by DNA electrophoresis.  These results demonstrate that even after completely losingcultivability, it is possible to find live cells in drinking water in theVBNC state. This suggests it is necessary to employ new methods todetect pathogens in water after disinfection to guarantee a safedistribution of drinking water.P.ENV.55 Chromobacterium violaceum, Pseudomonas aeruginosa andRalstonia solanacearum: homologous fucose/mannose andheterologous fucose/mannose and galactose specific lectins Gilboa-Garber N1, Imberty A2, Sudakevitz D1, Zinger-Yosovich KD1and Garber NC11Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900Israel 2CERMAV-CNRS Joseph Fourier University, Grenoble, FranceP. aeruginosa (PA), R. solanacearum (RS) and C. violaceum (CV),which are soil- and water-borne saprophytic bacteria, occasionallybecoming aggressive opportunistic pathogens (PA and CV mainlyattacking animals whereas RS damages plants), differ in theirmetabolites. PA produces a soluble green pigment pyocyanin and CV -a violet insoluble violacein formation. However, they display aconsiderable genomic similarity, encompassing their fucose/mannose-binding lectins: PA-IIL (1977), RS-IIL (2004) and CV-IIL (2005). Inaddition, PA produces a unique galactophilic lectin, PA-IL and RS – afucose>mannose binding lectin, RSL, that resembles PA-IIL in itssugar specificity while being similar to the soil fungus Aleuria aurantiain its structure (Wimmerova et al., JBC, 2003). These lectins arecoproduced with the bacterial virulence factors under Quorum Sensingcontrol and their level is elevated in osmoprotectant presence. Theirrole might be associated with selective adhesion of the bacteria totarget cells, enabling aggressive enzymatic attack on them. Hence thefive isolated pure lectins are very useful for analyses of animal andplant glycans and as probes for glycosylated compounds, especiallythose aimed to be useful for blocking the homing and adhesion of thepathogenic bacteria to the target cells. P.ENV.56 Strain N64, an exopolysaccharide-producing bacteria capable ofdenitrifyingGonzález-Domenech, C.M., Martínez-Checa, F., Béjar, V. andQuesada, E.Department of Microbiology. University of Granada. Campus ofCartuja, s/n. 18071. Granada. Spain.cmgodo@ugr.es Strain N64 is an halophilic, exopolysaccharide-producing anddenitrifying bacterium, isolated from a hypersaline soil, bordering asaline saltern on the Mediterranean coast in Almería (Spain). StrainN64 is Gram-negative, non-motile and shows a morphology of shortrods, appearing single or associated; it is oxidase and catalase positive.Phylogenetic analyses (sequencing of 16S DNAr gene) indicates thatthe strain in question belongs to the genus Halomonas. Thisinformation has been complemented by sequencing of the narH, nirSand nosZ genes, keys in the denitrification process and useful toinvestigate phylogenetic relationships among communities ofdenitrifying bacteria. The G+C content is 67.14% mol. Phenotypictests also show that it could be assigned to this genus: It is moderatelyhalophilic, growing best with 5%-10% w/v of salts. It is alsochemoheterotrophic, growing with different carbohydrates, organicacids, alcohols or aminoacids as sole sources of carbon, nitrogen andenergy. It is able to grow in a wide range of temperatures, from 4ºC to45ºC. It hydrolyses caseine, tyrosine and esculine. Urease, lecithinase,phenilalanine deaminase and phosphatase are positive. Growth inMcConkey agar is also positive. The strain N64 can grow inanaerobiosis with nitrate or fumarate as terminal electron acceptors.The results obtained suggest that strain N64 could belong to the genusHalomonas. P.ENV.49 Chromium effects on bacteria respirationR.Francisco1, A. Moreno2 , M.C. Alpoim2 and P.V. Morais21Instituto do Ambiente e Vida, 3004-517 Coimbra, Portugal. 2Departamento de Bioquímica, Faculdade de Ciências e Tecnologiada Universidade de Coimbra, Apartado 3126, 3001-401 Coimbra,Portugal.The present work focuses on the effect of Cr(VI) on cell respiration ofstrains Ochrobactrum tritici strain 5bvl1 and Microbacterium sp. strain3a, previously isolated from a contaminated wastewater treatment plantand both considered resistant and able to reduce this metal with acytosolic NAD(P)H-dependent enzyme. Cr(VI) was tested both aschromate and dichromate salts. Cr(III) effect on respiration were alsoassessed, and the experiments were also carried on filogenetically closeCr-sensible bacteria for control purposes, namely type strain O. triticiLMG 18957 and Microbacterium sp. strain CV17a.P.ENV.50 Characterization of metal resistance of yeasts isolated fromextremely acidic and metal rich environments in the São Domingospyrite mine (Portugal)Gadanho M and Tenreiro RUniversidade de Lisboa, Faculdade de Ciências, Centro de Genética eBiologia Molecular and Instituto de Ciência Aplicada e Tecnologia,Edifício ICAT, Campus da FCUL, Campo Grande, 1749-016 Lisboa,PortugalIn the present work a total of 19 yeast strains were studied. Amongthese, 18 (representing at least 10 species) were obtained from threeextreme acidic and metal rich aquatic environments located in thePortuguese side of the Iberian pyrite belt (IPB). In addition, a culturecollection strain of Saccharomyces cerevisiae (PYCC 3507) wasincluded. The minimum inhibitory concentration of seven metals (assulfates) and the lowest pH where growth occurred were determinedfor each individual strain and as consortia. All the experiments wereperformed in liquid medium using an automated 100-well plate readerBioscreen C. The highest metal concentration resistance recordedwere: >85 g/L Fe2+, 60 g/L Zn2+, 34 g/L Mg2+, 14 g/L Cu2+, 9 g/LNi2+, 4 g/L Co2+ and 2 g/L Cd2+. The most polymetallic resistantstrain belong to the ascomycetous genus Lecythophora. Amongbasidiomycetous yeasts, several Cryptococcus spp. strains presentedthe highest resistance levels. On the other hand, a higher resistance wasobserved when more acidic conditions were used for growth,highlighting the influence of pH in metal resistance. Correlationbetween resistance profiles and incidence/abundance of the yeastspecies in the sampled sites indicates that several of these yeasts areautochthonous to the extreme environment of the IPB.P.ENV.51 Culturable bacterial diversity in the drinking water distributionsystem of SevillaGallego, V *, Garcia M T and Ventosa ADept. Microbiology and Parasitology, Faculty of Pharmacy, Universityof Sevilla, 41012 Sevilla, Spain The present study is focused on monitoring the biodiversity of drinkingwater of  Sevilla (Spain) by measuring several physico-chemicalparameters (temperature, pH, free chlorine, total chlorine, total organiccarbon), total and viable cell counts and heterotrophic plate count.Moreover, a broad study of the culturable bacteria of this system wascarried out based on molecular and phylogenetic analysis of 16S rRNAgene sequences. For this purpose, four sampling campaigns during oneyear were carried out. A total of 235 strains were isolated and studiedbased on their partial 16S rRNA gene sequences. They were affiliatedto different phyla but only 224 were related to a specific genus. Theculturable bacteria mainly belonged to the a-Proteobacteria (62%), g-Proteobacteria (18%) and Firmicutes (12%). Actinobacteria (3%), b-Proteobacteria (3%) and Bacteroidetes (2%) were also detected. Thegenera Methylobacterium, Sphingomonas and Bacillus werepredominant but other 37 genera were present too. 16S rRNA sequencesimilarities of some strains were lower to 97% with respect to bacterialspecies and they were characterized by using a polyphasic taxonomicapproach. Until now, five new species of the genus Methylobacterium,one Pedobacter species, one Massilia species, one Chryseobacteriumspecies and one Roseomonas species have been described. Currently,28 strains are under research to ascertain their taxonomic affiliation asnew species or even as new genera.P.ENV.52 Effect of conditioning on the bacteriological quality of breedersand larvae in a hatchery of Ostrea edulis L. in the Northwest ofSpainCarballas C.G., Piñeiro-Vidal M., Muñoz A., Santos Y.Dpto. Microbiología y Parasitología, Facultad de Biología,Universidad de Santiago de Compostela. 15782 Santiago de Compostela, Spain mpcarba@usc.esGalicia (Northwest of Spain) has a long tradition in the production andconsumption of oyster. However, the culture of Ostrea edulis decreaseddramatically in 80’s probably due to the introduction of foreign oysterwithout the required sanitary controls. Currently, Galician producersdepend on oyster seed importation for their growing. An alternative forthe oyster industry could be the production of oyster seed in hatchery. The aim of this work was to study the bacteria associated to Ostreaedulis breeders maintained at different photoperiod and temperatureconditions and their effect on larval culture success. With this aim,oysters harvested from the wild have been used. Shell were stacked infibre tanks, seawater was then added and heated to temperatures thatenhance the obtention of mature oyster breeders. In order to determinethe microbiological quality of breeders, microalgae used for feeding aswell as the water of culture system, samples were periodicallycollected since the reception of breeders in hatchery until gonadmaturity was achieved. The samples were inoculated in general andselective culture media. Little differences were observed in thebacteriological levels of the analyzed samples regardless of thetemperature and photoperiod used to induce oyster maturation.

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Survival of VBNC legionella pneumophila in drinking water following chlorine disinfection

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Universidade do Minho: RepositoriUM