S and genotypes is the 1st step of correlation of these data with antimicrobial resistance profiles.Materials AND METHODSBacterial strains. A collection of 1,602 S. aureus strains collected in 2002 to 2003 from different geographical origins, representing both hospitaland communityacquired infections and hosted at the strain collection of Quotient Bioresearch (Fordham, United kingdom), was investigated. The same strain collection had previously been screened for susceptibility to the biocide triclosan (22). S. aureus strains employed for in vitro mutant choice integrated the biocide reference strain ATCC 6538, the standard laboratory strain RN4220, the classical reference strain for antimicrobial susceptibility testing, ATCC 2593, and three methicillinresistant S. aureus (MRSA) strains with sequenced genomes, MW2, COL, and Mu50, of which the latter two harbor plasmids containing the quaternary ammonium compound resistance gene (qacA) (22). Chemical agents. Compounds used were ethidium bromide (EB; ten mg/ml; Fluka, Steinheim, Germany), ciprofloxacin (CIP; two mg/ml; Bayer, Leverkusen, Germany), norfloxacin (NOR; 50 mg/ml in acetic acid; N9890; Sigma, Steinheim, Germany), benzalkonium chloride (BZC; 100 mg/ml in water; B6295; Sigma), chlorhexidine digluconate (CHX; 100 mg/ml in water; C9394; Sigma), and acriflavine (AF; 100 mg/ml in dimethylsulfoxide [DMSO]; A8126; Sigma). Susceptibility testing. MICs have been determined using the broth microdilution system (28). Minimum bactericidal concentrations (MBCs) were determined by subculturing 10 l from each and every nicely with out visible bacterial growth on MuellerHinton agar plates. Soon after 24 h of incubation at 37 , the dilution yielding 3 colonies or fewer was scored as the MBC, as described by the CLSI for starting inocula of 1 105 CFU/ml (29). Reference strains had been included in all 105 MIC and MBC determinations, and we confirmed the reliability from the susceptibility tests for the biocidal compounds by evidencing deviations in the imply final results of only a single dilution.1256822-12-4 structure MLST analysis.5,6-Diiodobenzo[d][1,3]dioxole custom synthesis Multilocus sequence typing (MLST) was performed on a group of 91 clinical isolates carrying qac determinants as described previously (30).PMID:23357584 The allelic quantity and STs were assigned using the S. aureus MLST database (http://saureus.mlst.net), even though the clustering of associated STs, defined as clonal complexes (CCs), was analyzed with all the BURST algorithm (http://eburst.mlst.net). New alleles and STs have been submitted towards the S. aureus MLST database. Activity testing. Benzalkonium chloride and chlorhexidine activity testing against the reference S. aureus strain and chosen isolates have been performed by following EN 1276 (31). Briefly, 1 ml of a test suspension of microorganisms at a concentration in between 1.five 108 and 5CFU/ml was mixed with albumin from the bovine serum Cohn V fraction (A2153; Sigma) at a concentration of 0.03 g/liter. Immediately after two min, 8 ml with the test solution answer was added and mixed. Test solution solutions have been obtained by diluting benzalkonium chloride (B6295; Sigma) and chlorhexidine digluconate (C9394; Sigma) in challenging water (119 mg/liter MgCl2, 277 mg/liter CaCl2, and 280 mg/liter NaHCO3). The mixture was maintained at 20 ( 1 ) inside the test tube for five min ( ten s). Just after this get in touch with time, a 1ml aliquot from the test tube was transferred to a tube containing eight ml on the neutralizer (3 g/liter lecithin, 30 g/liter polysorbate80, five g/liter sodium thiosulfate, 1 g/liter Lhistidine, and 30 g/liter saponin in diluent) an.
