Optimization of processing conditions for the quantification of enterococci biofilms using microtitre-plates.

We performed a comparison of four selected protocols from the literature using microtitre-plate and colorimetric biomass assay for evaluation of enterococci biofilm formation ability and optimized a protocol after the identification and stringent performance of biofilm formation steps. The optimized protocol uses a dynamic model that provided a greater discrimination of enterococci biofilm formation ability, and could better simulate in vivo real conditions. Moreover different biofilm quantification approaches, such as the colorimetric biomass (crystal violet), the resazurin and CFU's assays could be used with the optimized protocol, with adequate reproducibility. This study also recognizes that parameters such as the biofilm formation index (BFi), the cut-off values and the Z' factor provide greater accuracy, possibility of inter and intra laboratory comparison and quality evaluation of the biofilm screening assays, respectively.

Molecular epidemiology of Pseudomonas aeruginosa clinical isolates from Portuguese Central Hospital.

The relatedness between clinical isolates of P. aeruginosa obtained from patients during their stay in a Portuguese Central Hospital was evaluated. Genotypic fingerprinting (M13-PCR), phenotypic methods (biotyping and antibiotyping) and epidemiological information (spatial and temporal links) were used to evaluate the relatedness between 88 clinical isolates (68 patients), selected randomly out of 189. Sixty-two M13 types were found, 12 of them containing isolates from more than one patient. Thirty-four antibiotypes were found, as well as a significant association (p < 0.05) between epidemic isolates and multiresistance patterns. The nosocomial transmission of P. aeruginosa strains may be limited since M13 typing demonstrated a high degree of diversity among all the isolates, suggesting the occurrence of mainly independent infectious episodes. The results show the possible occurrence of cross-acquisition, cross-colonization and cross-infection and suggest an epidemic population structure for P. aeruginosa in this hospital.

Effect of subinhibitory concentration of piperacillin/tazobactam on Pseudomonas aeruginosa.

Subinhibitory concentrations (sub-MICs) of antibiotics, although not able to kill bacteria, can modify their physico-chemical characteristics and the architecture of their outermost surface and may interfere with some bacterial functions. This study investigated the ability of sub-MIC piperacillin/tazobactam (P/T) to interfere with the bacterial virulence parameters of adhesiveness, cell-surface hydrophobicity, motility, biofilm formation and sensitivity to oxidative stress. Antimicrobial activity against five Pseudomonas aeruginosa clinical isolates, representative of clonal lineages of 96 strains of nosocomial origin, and six control strains (ATCC 27853, PAO1, AK1, MT1562, PT623, PAO1algC) was evaluated in vitro using the NCCLS microdilution method. The effects of sub-MIC on bacterial adhesion and biofilm formation were studied using a modified microtitre plate assay. The relative cell-surface hydrophobicity of P. aeruginosa strains was determined by measuring their ability to adhere to n-hexadecane. P. aeruginosa that had been exposed overnight to P/T and incubated with P/T in the plate were also screened for their ability to swim using flagella and to twitch and for their sensitivity to oxidative stress. The results obtained showed that the impact of sub-MIC P/T on bacterial characteristics was different for the various strains of P. aeruginosa. There was a change in bacterial morphology and hydrophobicity that could explain a significant decrease in adhesion values in all clinical isolates and controls tested, a decrease in biofilm formation, a significant increase in sensitivity to oxidative stress, a significant decrease in flagellum-mediated swimming and a decrease in type IV fimbriae-mediated twitching. The results obtained indicate that sub-MIC P/T interferes with the pathogenic potential of P. aeruginosa.