부산대학교 도서관

Ventilator-associated pneumonia (VAP) is defined as a hospital-acquired pneumonia commonly occurring in intensive care units in a patient after 48h of mechanical ventilation via an endotracheal tube (ETT) or tracheostomy tube. It is one of the most common infectious complications in critically ill patients. The prevalence of VAP varies between 9-65% and mortality rates are high (15- 76%). It has been reported that microbial biofilms cover the ETT after 7-10 days depending on the in vitro method of assessment used. A number of microorganisms play a dominant role in VAP, many of which can form biofilms on ETT surfaces, including Klebsiella pneumoniae. The presence of Candida spp. in respiratory secretions of patients with VAP has been associated with longer mechanical ventilation, prolonged stay and worse outcomes. Furthermore, colonisation of ETT by Candida species, principally, Candida albicans, has been associated with the development of VAP caused by a number of bacteria. The present research was designed to further analyse the relationship between Klebsiella species and C. albicans within dual species biofilm in in vitro VAP model. Klebsiella spp. was chosen as its emergency of resistance is increasing in ventilated patients. Antimicrobial effects of meropenem (MEM) and fluconazole (FLC) were examined against single and dual-species biofilms. Four clinically paired Klebsiella spp. and C. albicans were used. Single and dual-species biofilms were grown in 24-well plates for up to 72h. Biofilms were exposed to MEM and/or FLC at different concentrations (0.4, 4.0 and 400 mg/L, reflecting sub, peri and supra-MIC concentrations) for 24h. Then, biofilms were analysed for cell viability using culture (CFU/mL). A qPCR molecular method based on propidium monoazide (PMA) was used in qPCR to aid live/dead discrimination. The metabolic activities of the biofilms were measured by using the 2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-5- [(phenylamino) carbonyl]-2H-tetrazolium hydroxide colorimetric assay (XTT). Electron microscopy (EM) and confocal laser scanning microscopy (CLSM) were used to visualise any changes in biofilms structure following treatment. In vitro biofilms revealed that all isolates in single and co-culture conditions showed a gradual increase in the number of cells and metabolic activity over time, reaching peak values between 72 h and 96 h. Supra-MIC concentrations of both MEM and FLC were effective in reducing biofilm viability and metabolic activity of both isolates. In contrast, sub-MIC and peri-MIC concentrations of FLC and MEM increased the metabolic activities and viability of the biofilms. Our results suggest that treating dual-species biofilms associated with VAP with sub-MIC or peri-MIC concentrations of MEM and FLC may allow bacteria and fungi to form barriers against the antimicrobial agents in form of mature biofilms.