Infection, Methods and Techniques, 00520, General biology – Symposia, transactions and proceedings, 12512, Pathology – Therapy, 25502, Development and Embryology -, General and descriptive, 31000, Physiology and biochemistry of, bacteria, 36001, Medical and clinical microbiology – General and, methods, 38504, Chemotherapy – Antibacterial agents, Gram-Positive Cocci, Eubacteria, Bacteria, Microorganisms, Bacteria,, Eubacteria, Microorganisms, Micrococcaceae , [Staphylococcus, aureus, (pathogen)] [Staphylococcus epidermidis, (pathogen)]/Rodentia,, Mammalia, Vertebrata, Chordata, Animalia, Animals, Chordates, Mammals,, Nonhuman Vertebrates, Nonhuman Mammals, Rodents, Vertebrates, Muridae, , [mouse, (host)]/Gram-Negative Aerobic Rods and Cocci,, Eubacteria, Bacteria, Microorganisms, Bacteria, Eubacteria,, Microorganisms, Pseudomonadaceae , [Pseudomonas aeruginosa, (pathogen)], Teflon catheter, medical equipment/antibiotic therapy, therapeutic, method/bioluminescence, analytical method/medical devices biofilm, monitoring, analytical method/medical implants, medical, equipment/rapid, direct non destructive, real time quantitative, monitoring methods, development, monitoring method IVIS, XenogenAbstract :
Background: Microbial adhesion and biofilm formation on medical implants is a common occurrence and represents a serious medical problem, as these bacteria are difficult to eradicate with antibiotic therapy and leads to chronic infections. Rapid, direct, non destructive, real time quantitative monitoring methods that are adaptable to the clinical situation are needed to develop new preventive and therapeutic methods to combat biofilm related infections. Method: We have developed a rapid, innocuous method for real time monitoring of biofilms in vitro and in a mouse infection model by establishing biofilms of bioluminescent bacteria on Teflon catheters. The capacity of the microbes to form biofilm on catheter material, their suitability for long-term experiments, and metabolic state of biofilms was assessed in situ by monitoring the bioluminescence with an IVIS imaging system (Xenogen Corp., Alameda, CA). Results: Three different biofilm forming bacterial pathogens, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, made bioluminescent by insertion of the lux operon, produced significant bioluminescent signals, both in vitro and in mice, allowing effective assessment of the physiological state of biofilm. Unlike plasmid-based lux constructs, chromosomal lux gene integrants were stable, and bioluminescence detectable for 21 days. Viable cell counts and light output were parallel, highly correlated (r=0.95) and could be maintained in vitro for 15 days or longer if growth medium is replenished every 24h. For the complete removal of bacteria from support surface to enumerate viable cells using conventional methods, vigorous votexing (5 min or more) was necessary. Aged biofilms (24 h) of P. aeruginosa were the most difficult to disassociate from the catheter followed by S. aureus. Conclusion: The ability to monitor biofilms in real time without exogenous sampling accelerates the study of biofilm population. Since the metabolic activity of viable cells could be detected directly on support matrix non destructively and non invasively, the methodology is especially appealing for in vivo and drug development studies.