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Background The 2010 European Antimicrobial Surveillance System Annual report showed high proportions of resistance, despite of the situation appears generally stable in Europe with few countries reporting significant increasing (Malta and France) or decreasing (Greece, Italy, Germany) trends to different antimicrobial agents. High proportions of Pseudomonas aeruginosa resistance were reported by many countries especially in southern and eastern Europe. Methods A cross-sectional study was carried on to compare the antibiotic resistance of P. aeruginosa towards all the other Non Fermentative Gram Negative Bacteria (NFGNB) isolated from different aqueous sources in generic and high-risk wards of a Teaching Hospital in Rome. Municipal water, dyalisate, fluid for the bronchoscopes disinfection effectiveness assay, double osmosis treated water samples were routinely collected from September 2004 to September 2010. P. aeruginosa was identified by growth characteristics and biochemical fermentation tests; the antimicrobial susceptibility was automatically evaluated with ATB PSE 5 strips (BioMerieux). The chi-squared test (χ2) was performed to investigate the differences of the resistance between P. aeruginosa isolates versus the other NFGNB; the statistical significance was set at p <0.05. Results Out of 2016 samples, 116 strains were isolated, 40 of them (34.5%) were P. aeruginosa. Among the 19 tested antibiotics, significant differences in resistance (p<0.001) between the two bacterial groups were observed for: amikacina (χ2= 18.56), ampicillin-sulbactam (χ2=25.37), colistina (χ2 = 40.65), cotrimoxazole (χ2 = 54.01), gentamicine (χ2 = 25.72) and Tobramicin (χ2 = 23.17). Conclusions Our results suggest a restraint of P. aeruginosa resistance to the new antimicrobial agents tested, such as carbapenems, fluoroquinolones and cephalosporins, if compared to all the other NFGNB resistance, suggesting judicious and prudent use of new drugs. The understanding of the ecological rol