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The objectives of this study were to: investigate the presence of bacteria of probable hospital origin in water, sludge, sediments andmussels from shellfish cultivation areas at the mouth of the Ulla River on the southern coast ofGalicia (Ria de Arousa);study the incidence of multiresistant microorganisms in the effluents of Santiago de Compostela University Hospital; evaluate the capacity of multiresistant microorganisms of hospital origin to cross the barrier ofthe Silvouta wastewater treatment plant (WWTP) in Santiago de Compostela and theiraccumulation in sludge and soil amended with sludge; and, analyze the genotype of multiresistant microorganisms isolated from different types of samples(wastewater, surface water, seawater, sludge, sediments and mussels) to establish theepidemiologic relation betweenmicroorganisms present in effluent of the Santiago de Compostela University Hospital, andeffluent of the WWTP and shellfish cultivation areas at the mouth of the Ulla River on thesouthern coast of Galicia (Ria de Arousa). The count of E. coli resistant to multiple antibiotics will be carried out using the following procedures:water samples from the Tambre and Sar rivers and wastewaters: membrane filtration technique andinoculation of filters on Coli-ID agar (bioMerieux, France) at 36C/24 h (Araujo et al. 2001); sludgesamples from WWPT and soil amended with sludges - homogenization in a sterile blender (50 gsludge in 450 mL phosphate buffer); and, after 30 minutes of sedimentation, the upper liquid phase andserial tenfold dilutions of this upper liquid phase will be poured onto Coli-ID agar and incubated at36C/24 h (Araujo et al. 2002).In each case, the Coli-ID agar will be supplemented with 4 widely used antibiotics. Furthermore, totalE. coli count will be made in the same medium without supplementation.The same procedures described for E. coli in three types of samples will be used for the multiresistantAcinetobacter spp count. In this case, Baumann agar with and without antibiotics and incubated at30C/48h will be used as the initial medium for culture and isolation of Acinetobacter spp(Guardabassia et al. 2002). Bacterial strains growing on Baumann agar will be identified asAcinetobacter spp. Culture isolates will be confirmed as Acinetobacter spp using molecular biologytechniques based on the polymerase chain reaction (PCR), which is highly specific for microorganismconfirmation in a short period of time, about 4 hours after culture isolation. The fact thatculture isolated microorganisms were available simplified PCR confirmation by enhancing detectionsensibility.From each sample, a representative number of bacterial strains growing on culture media wasrandomly isolated and identified. Antimicrobial susceptibility testing of isolates was performed withthe ATB system (bioMerieux, France) using 12 antimicrobial agents representative of majorantimicrobial classes. Also, the minimal inhibitory concentration (MIC) was determined according tomethods recommended by the National Committee for Clinical Laboratory Standards (NCCLS 2002Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 3rd Edition.Approved Standard M7-A5. Wayne.Pa. USA).The presence of antibiotic genes in E. coli isolates was analyzed by PCR and PCR-restrictionfragment length polymorphism analysis and sequencing. Includes 14 references, table, figures.

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Edition: Vol. - No. Published: 11/01/2006 Number of Pages: 13File Size: 1 file , 510 KB