STANDARDIZED  TECHNIQUE FOR DETECTION OF AEROMONAS HYDROPHILA IN TREATED WASTE WATER SAMPLES

Danielle Escudeiro (FSP-USP); Livia Balsalobre (FSP-USP); Patricia Souza (FSP-USP); Milena Dropa (FSP-USP); Glavur Rogério Matté (FSP-USP); Maria Inês Sato (CETESB); Maria Elayse Hachich (CETESB); Maria Helena Matté (FSP-USP)

Aeromonas spp. are ubiquitous bacteria found in a variety of aquatic environments worldwide, including bottled water, chlorinated water, well water, and heavily polluted waters. While originally thought to be an opportunistic pathogen in immunocompromised humans, an increasing number of cases of intestinal and extraintestinal disease documented worldwide suggest that it is an emerging human pathogen. The organism is included in the Contaminant Candidate List by the Environmental Protection Agency, and U.S. water supplies are routinely examined for it. The aim of this study was to standardize a technique to detect Aeromonas hydrophila in treated waste water samples, using filtering membrane, specific isolation medium and specie-specific primers. One liter of treated waste water was collected from Sewage Treatment Plants. A serial dilution to 10^-10 was carried out, each dilution was filtered using a 0,45mm membrane and placed onto a plate containing Aeromonas isolation Agar (Base) medium and incubated at 35ºC for 24h. After incubation plates were searched for dark green colonies, the membrane dilution containing from 30 to 300 characteristics colonies was chosen to represent the sample. Colonies were screened in Kliger Iron Agar. Non-fermented, H2S positive or negative oxidase isolates were discarded. The selected isolates were heat-freeze extraction and molecular specie confirmation was performed using specie-specific primers for Aeromonas hydrophila, using a conserved region of 16S rDNA gene. The presence of Aeromonas hydrophila was observed in the sample collected using the methods described above. The amplified fragments were sequenced. Based on the results presented one can conclude that the filtering technique associated with the serial dilution is a fast and easy method for detection of A. hydrophila in water samples. Also the specie-specific primers showed highly specificity towards A. hydrophila isolates. Regarding the Aeromonas isolation Agar (Base) medium, it was noticed that the medium plate can contain dark green colonies that do not represent A. hydrophila strains. One can also conclude that treated waste water samples may contain A. hydrophila isolates, representing a threat for public health, since these organisms are considered emerging pathogens carrying virulence and resistance factors. The present work also highlights the importance of monitoring Aeromonas hydrophila in treated waste water. This work was supported by grants of FINEP (Financiadora de Estudos e Projetos).