Six different P. aeruginosa strains were isolated throughout the outbreak investigation, as previously reported . P. aeruginosa strain L2 was isolated in the triclosan-containing soap dispenser and isolates L3 and L4 from water outlets in patients rooms (environmental isolates). P. aeruginosa isolates 10, 11 and 13 were isolated from patients' biological specimens (clinical isolates). Isolates L2, 10, 11 and 13 all belonged to sequence type 175 and were indistinguishable by pulsotyping, RAPD and MLST analysis, while isolates L3 and L4 showed a distinct type . After primary isolation, isolates were sub-cultured for 4-6 times in Mueller-Hinton (MH, Oxoid, Milan, Italy) agar, and then stored frozen in MH broth.
Two commercial disinfectants were used in this study, designated product A and product B, whose composition (% w/v in water) is: (A) 0.5% 2,4,4'-trichloro-2'-hydroxy-diphenyl-ether (triclosan), 10% sodium lauryl-myristyl ether sulfate, 3% coconut glycerol, 2.5%coconut amido propyl betaine, and 1.5% alcohol ethoxylate; (B) 4% chlorhexidine gluconate, 15% polysorbate 20, 5% coconut diethanolamide, 4% alkyl polyglucoside, 1% polyethylene glycol (PEG)-150, 1% isopropyl alcohol, 0.1% essence, and 0.005% E 124.
Susceptibility tests for disinfectants
Taking into account the very poor solubility of triclosan in aqueous solutions , the antimicrobial susceptibility tests and the time kill assay were performed using the commercial soap formulations. In the commercial product A, triclosan is well soluble (it is 5,000 mg/L), and its addition to MH broth (that was adequately concentrated to have a final concentration of 1 × MH broth in all assays) did not result in any precipitation of the disinfectant.
P. aeruginosa was grown to the early stationary phase in MH broth. Bacterial cultures were diluted to 0.5 MacFarland turbidity in MH broth (~5 × 108 CFU/ml). This suspension was diluted 1:10 (~5 × 107 CF/mL) in MH broth, and 10 μl were used as inoculum in 96-well microtiter plates (Costar, Cambridge, Massachusetts) containing 190 μl of serial two-fold dilutions of the disinfectant soaps in 1 × MH broth. To test the highest concentration of triclosan (4,250 mg/L, which corresponds to the minimum soap dilution), 20 μl of 10 × concentrated MH broth were mixed with 170 μl of undiluted triclosan soap (product A) containing 5,000 mg/L triclosan and 10 μl of bacterial inoculum (~2.5 × 106). All other dilutions were prepared by combining appropriate volumes of 10 × concentrated MH broth, triclosan soap, sterile water, and the standard 10-μl bacterial inoculum.
To test the susceptibility of P. aeruginosa to chlorexidine digluconate (product B), standard two-fold dilutions of the disinfectant were generated in 1 × MH broth and inoculated with ~2.5 × 106 CFU/mL in 96-well microtiter plates (Costar).
Since the disinfectant soaps were commercial mixtures of triclosan or chlorhexidine with other ingredients which may or may not be inert, the observed minimal inhibitory concentrations (MICs) were called "apparent MICs" (a-MICs). The a-MIC was expressed as the minimal concentration of the disinfectant in the commercial formulation causing no growth after 48-h incubation at 37°C [12, 13], with chlorhexidine > 50 mg/L  and triclosan > 128 mg/L  as resistance breakpoints. Each experiment was performed in triplicate.
Determination of the bactericidal activity for disinfectants
P. aeruginosa L2 suspensions (~5 × 107 CFU/mL) were made in MH broth supplemented with either product A (triclosan range: 1,062-4,250 mg/L) or product B (chlorhexidine digluconate range: 12.5-1,250 mg/L), and incubated at 37°C. The two products were diluted in MH broth, prepared at the appropriate concentration in order to compensate for the addition of an appropriate volume of disinfectant and a constant bacterial inoculum. At defined intervals, aliquots were neutralized by rapid serial dilution in MH broth supplemented with 0.5% lecithin and 4% polysorbate 20 , plated on MH agar plates, and examined after 24-h incubation at 37°C for determination of viable counts.
Adaptation to high triclosan concentration
The frozen stock of P. aeruginosa L2 was readapted to triclosan by passages in gradually increasing triclosan concentrations. Inocula of ~5 × 105 CFU/mL were serially cultured in stepwise increasing triclosan concentrations in MH broth at 37°C, starting with 1/2 the MIC and doubling the concentration at each passage until evidence of full growth.
Antimicrobial susceptibility testing
The susceptibility of triclosan-adapted and unadapted P. aeruginosa to a panel of antibiotics which are typically exported by RND efflux pumps , namely tetracycline, ciprofloxacin, amikacin, levofloxacin, carbenicillin and chloramphenicol (all from Sigma-Aldrich) was determined by the broth microdilution method according to the CLSI guidelines  either with or without the RND efflux pump inhibitor phenyl-arginine-β-naphthylamide (PAβN) or the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Sigma-Aldrich) at the predetermined concentrations of 50 μM and 100 μM, respectively. Each experiment was performed in triplicate.