Most of the antibiotic susceptibility tests performed in this study were performed with P. aeruginosa PAO1 colony biofilms that were grown for either 4 or 48 h prior to antibiotic exposure. The polycarbonate membranes that supported colony biofilms were initially seeded with approximately 106 CFU of cells. Bacteria began to grow exponentially and remained in exponential phase for approximately 6 h (Fig. ). The specific growth rate calculated between 0 and 5 h was 1.14 ± 0.03 h−1. Growth slowed after 6 h, and the colony appeared to enter stationary phase. The average specific growth rate between 24 and 48 h was 0.02 ± 0.01 h−1.
FIG. 1. Growth of colony biofilms under aerobic conditions (solid circles), anaerobic conditions (empty circles), and anaerobic conditions with nitrate (gray circles). All colony biofilms were grown under aerobic conditions for the first 4 h and were then transferred (more ...)
The antibiotic susceptibilities of mature (48-h-old) colony biofilms were investigated in vitro. Bacteria were challenged with approximately 10 to 20 times the MIC measured under aerobic conditions (Table ) for 12 h. The antibiotics killed the P. aeruginosa organisms growing in 48-h-old colony biofilms poorly (Table ). The largest log reduction was recorded with ciprofloxacin (log reduction, 1.13). All of the other antibiotics generated log reductions of 0.82 or less for mature biofilms.
MICs of antibiotics for P. aeruginosa under various conditions of oxygen and nitrate availability
Susceptibilities of mature (48-h-old) colony biofilms to antibiotics
A direct measurement of the oxygen concentration profile in 48-h-old P. aeruginosa colony biofilms was obtained with oxygen microelectrodes (Fig. ). Mature biofilms were mostly anaerobic. These measurements show that oxygen penetrated approximately 50 μm into the colony from the air interface. As the colony biofilms were approximately 210 μm thick, the oxygen-replete portion can be estimated to be about one-quarter of the entire colony. Most of the mature colony biofilm can therefore be assumed to inhabit a state of anoxia or a state with low dissolved oxygen concentrations.
Oxygen concentrations in mature (48-h-old) P. aeruginosa colony biofilms (solid circles) versus sterile agar (empty circles). The result shown is the average for three oxygen concentration profiles.
When 48-h-old colony biofilms formed by a strain of P. aeruginosa carrying an inducible, stable GFP were exposed to the inducing agent IPTG for 4 h, only those bacteria in a narrow band adjacent to the air interface were able to express GFP (Fig. ). In Fig. , green defines the region of the biofilm in which active protein synthesis is taking place, while red indicates biomass that is inactive. It can be deduced that IPTG penetrates the biofilm because the zone of active GFP induction is at the edge of the biofilm opposite from that to which the inducer was delivered. In other work (Werner et al., submitted), it has been shown that the zone of GFP induction corresponds to the region of bacterial growth, as mapped by other techniques. Because the activation of GFP fluorescence is oxygen dependent, the bright zone could reveal only the region of aerobic protein synthesis. In this experiment, however, in which neither an alternative electron acceptor nor arginine was present, the zone of aerobic metabolism is predicted to be the only zone of active protein synthesis. The dimension of this zone of GFP synthesis was determined by image analysis to be 32 ± 3 μm. These results show that 48-h-old P. aeruginosa colony biofilms are physiologically heterogeneous. The majority of the biofilm occupied an inactive or anaerobic state.
FIG. 3. Pattern of protein synthesis in P. aeruginosa colony biofilms that were 48 h (A) or 4 h (B) old. Green corresponds to induced GFP and red derives from a counterstain for all biomass. The supporting membrane and agar were at the bottom and the air was (more ...)
Oxygen availability influenced the growth of young (4-h-old) colony biofilms. At this early stage of development, the bacteria in colony biofilms incubated in air still had access to oxygen and all of the cells were metabolically active. When a biofilm formed by the strain carrying the inducible GFP construct was grown for 4 h and then induced with IPTG for 4 h, the cells did not yet appear to be fully aggregated and they all expressed GFP (Fig. ). There were no red zones in the biofilm, as there were in the 48-h-old biofilms (Fig. ), which would have indicated inactive or anaerobic biomass. The bacteria in the 4-h-old biofilms were still growing. The average specific growth rate calculated between 6 and 16 h for colonies grown under aerobic conditions was 0.16 ± 0.01 h−1 (Fig. ). When 4-h-old colony biofilms were transferred to anaerobic growth conditions, growth was arrested within 2 h. The average specific growth rate under anaerobic conditions over the same time interval (4 to 16 h) was −0.04 ± 0.02 h−1. This shows that oxygen is important for colony biofilm growth from 4 to 16 h under the conditions tested.
Young (4-h-old) colony biofilms challenged under aerobic growth conditions were susceptible to antibiotics, especially ciprofloxacin and tobramycin (Fig. ). When the same assays were performed under anaerobic growth conditions, antibiotic efficacies were reduced in all cases (Fig. ). For example, 4-h-old colony biofilms challenged with ciprofloxacin and tobramycin on TSA in air experienced almost complete killing (log reductions, 5.05 ± 0.31 and 5.67 ± 0.00 [killing to the detection limit], respectively), while the log reductions resulting from the same two agents were much less (2.61 ± 0.13 and 2.14 ± 0.42, respectively) in the absence of oxygen. These differences were statistically significant (P < 0.001 in both cases). It was also determined that the other three antibiotics tested, carbenicillin, chloramphenicol, and tetracycline, had reduced efficacies under anaerobic growth conditions. These results show that oxygen availability is an important determinant of antibiotic susceptibility in P. aeruginosa.
FIG. 4. Effect of oxygen availability on antibiotic susceptibilities of young (4-h-old) P. aeruginosa colony biofilms. The error bars represent the standard errors of the means for each value. •, control; ○, ciprofloxacin; , tobramycin; (more ...)
Mature (48-h-old) colony biofilms may contain bacterial cells that have been deprived of oxygen for a prolonged period. To simulate the state of low metabolic activity that bacteria may occupy in mature biofilms, 4-h-old colony biofilms were incubated in an anaerobic environment for 24 h prior to antibiotic challenge. The subsequent 12-h antibiotic treatment was also delivered under anaerobic conditions. There was little difference between the log reductions measured in these experiments and those determined under anaerobic conditions without prolonged anoxia. The only exception was for ciprofloxacin, in which case the log reduction was reduced to 1.75 ± 0.08 under conditions of prolonged anaerobic incubation.
To assess the effects of nitrates on P. aeruginosa growth, 4-h-old colony biofilms were transferred to plates supplemented with 1% KNO3 and incubated both aerobically and anaerobically. The log increase in viable cell numbers over the 12-h test period in an aerobic environment was 1.38 ± 0.08, slightly less than that when no nitrate was present. On the other hand, colony biofilms that were incubated anaerobically grew more in the presence of nitrate than when nitrate was absent. The log increase for biofilms incubated anaerobically in the presence of nitrate was 0.87 ± 0.08 (Fig. ), which is statistically significantly larger than that for the anaerobic control without nitrate (P = 0.004). The specific growth rate in the presence of nitrates under anaerobic growth conditions calculated between 6 and 16 h was 0.36 ± 0.07 h−1. These results show that nitrate facilitates the growth of colony biofilms under anaerobic conditions but has a slight detrimental effect on aerobically growing biofilms.
The effect of nitrate supplementation on the antibiotic susceptibilities of 4-h-old colony biofilms was evaluated under both aerobic and anaerobic growth conditions. Nitrate slightly increased the MICs under aerobic conditions (Table ). The susceptibilities of young colony biofilms under aerobic conditions in the presence of nitrate were similar to the susceptibilities of these biofilms under aerobic conditions in the absence of nitrate for most antibiotics (Fig. ). For example, in the presence of oxygen, nitrate supplementation had no statistically significant effect on killing by ciprofloxacin (P = 0.42). The efficacy of killing by tobramycin decreased from a log reduction of 5.67 ± 0.00 (killing to the detection limit) in the absence of nitrate to a log reduction of 4.55 ± 0.56 in the presence of nitrate. The only antibiotic whose efficacy was notably altered by the addition of nitrate under aerobic conditions was carbenicillin (Fig. ). Carbenicillin had almost no effect on colony biofilm bacteria in the standard test in the presence of air and the absence of nitrate (log reduction, 0.34 ± 0.27). When nitrate was added, killing by this antibiotic rose to a log reduction of 4.04 ± 0.31. In summary, under aerobic conditions nitrate supplementation had little effect on the efficacies of most antibiotics, with the exception of carbenicillin, whose efficacy was substantially increased by the presence of nitrate.
FIG. 5. Effect of nitrate availability on antibiotic susceptibilities of young (4-h-old) P. aeruginosa colony biofilms under aerobic (A) and anaerobic (B) conditions. The error bars represent the standard errors of the means for each value. •, control; (more ...)
Under anaerobic conditions, nitrate supplementation decreased the levels of killing by ciprofloxacin and tobramycin (Fig. ). The log reductions obtained with both agents were more than halved in the presence of nitrate and the absence of oxygen, and these effects were statistically significant (P = 0.003 and P < 0.001 for ciprofloxacin and tobramycin, respectively). Nitrate had no statistically significant effect on the efficacies of the other three antibiotics under anaerobic conditions. The enhancement of the action of carbenicillin that was so striking under aerobic conditions was abolished under anaerobic conditions. These data show that nitrate can reduce the efficacies of clinically important antibiotics used for the treatment of P. aeruginosa infections when anoxia prevails.