Pathogenic characteristics of Pseudomonas aeruginosa strains resistant to carbapenems associated with biofilm formation

Background. In recent years the worldwide emergence of multidrug-resistant strains of Pseudomonas aeruginosa has been observed. This opportunistic pathogen produces mechanisms of resistance to several antibiotics. The resistance to carbapenems in P. aeruginosa strains has been associated with bacterial biofilm formation favored by the presence of exopolysaccharides (EPS) embedded in an extracellular matrix and to the production of type IV pili (T4P). We undertook this study to assess biofilm formation in clinical strains of P. aeruginosa resistant to carbapenems isolated at the Hospital Infantil de Mexico Federico Gomez (HIMFG) through quantification of totalreducing EPS and its association with the phenotypic expression of T4P. Methods. Antibiotic susceptibility tests were performed using the Kirby-Bauer method in 92 clinical isolates of P. aeruginosa; likewise, the minimum inhibitory concentration (MIC) was determined for imipenem (IMP) and meropenem (MEM) using the serial dilution method in agar plates with a Steers replicator. Production of metallo-b-lactamase (MBL) was determined by the disk diffusion method and synergism. Biofilm formation was performed in clinical isolates of P. aeruginosa resistant to carbapenems through the quantification of crystal violet, total sugar (anthrone), and reducing sugar (DNS), in addition to the phenotypic expression of T4P activity of twitching motility. The genetic diversity of biofilm-forming strains and producers of reducing sugars was evaluated by pulsed-field gel electrophoresis (PFGE). Results. There were 30.4% (28/92) of P. aeruginosa strains of pediatric origin and 50% (46/92) of urine samples that were recovered from the pediatric surgical ward. The results using the Kirby-Bauer method showed that >50% of P. aeruginosa strains were resistant to 12 different antibiotics. The MIC to carbapenems was 64 mg/mL, with 43.1% (25/58) for MEM and 56.8% (33/58) for IMP. Likewise, MBL production was observed in 43% (25/58) for MEM, 2% (1/58) for IMP, and 12% (7/58) for both. Qualitative and quantitative analysis showed that 82% (48/58) of P. aeruginosa strains resistant to carbapenems were high biofilm formers using the crystal violet method. From the high biofilm forming strains, 46.5% (27/58) showed concentrations of total EPS between 2000 and 6000 mg/mL and 27.5% (16/58) showed concentrations of reducing EPS between 316 and 1108 mg/mL. In addition, 75% (44/58) of these strains showed phenotypic activity of twitching motility. Conclusions. Detection of total sugars, reducing sugars, and the phenomenon of twitching motility are factors that promote the development of biofilms in clinical strains of P. aeruginosa resistant to carbepenems, which are also MBL producers. Our data suggest that these factors are involved in biofilm formation, which confer bacterium with the ability to survive, persist, and colonize its host.