Synergy of Silver Nanoparticles and Aztreonam against Pseudomonas aeruginosa PAO1 Biofilms

High-throughput biofilm antimicrobial susceptibility assay.To assess the antimicrobial properties of citrate-capped, dispersed AgNPs of different sizes (Fig. 1A), alone and in combination with the antibiotic aztreonam, we used the minimum biofilm eradication concentration (MBEC) high-throughput assay (29, 30, 41), which has been determined to reflect antimicrobial efficacy against biofilms more accurately than MIC assays (16). These assays involve specialized MBEC plate lids with pegs that support surface-attached bacterial growth (i.e., biofilms) and wells that support free-living (i.e., planktonic) populations. Sequential transfers of the plate lids containing the biofilms to different wells allow for the analysis to be separated into three distinct stages, i.e., growth, challenge, and recovery (Fig. 1B). This experimental design also permits determination of the MIC for planktonic cells and the MBEC within the same assay (42–44). The planktonic population of bacteria in the wells after the challenge-phase incubation (i.e., in the presence of antimicrobial) reflects the planktonic susceptibility of cells shed from the biofilm (42, 44) and is used to calculate the MIC; the planktonic population in the wells after the recovery-phase incubation (i.e., in fresh medium) indicates postchallenge biofilm viability (29, 30, 42, 43) and is used to calculate the MBEC (29, 30). Following the recovery incubation, the biofilm-covered pegs are stained with crystal violet to determine the biofilm biomass.

• Open in new tab
• Download powerpoint

FIG 1

High-throughput MBEC biofilm antimicrobial susceptibility assay. (A) Transmission electron micrographs of AgNPs (10 nm to 100 nm) used in this study. Bars, 100 nm. (B) Schematic illustration of the workflow used (i) to culture P. aeruginosa PAO1 planktonic and biofilm cultures (growth phase), (ii) to incubate biofilms in the presence of AgNP-aztreonam treatments and to determine MIC values for the planktonic cultures (challenge phase), and (iii) to determine biofilm biomass, viability, and MBEC values following incubation in fresh medium without antimicrobials (recovery phase) (see Materials and Methods for more details). (C) Reproducibility of biofilm formation, assessed in the growth phase of the experiment by plotting planktonic growth (based on planktonic cell density in the wells of the growth phase) versus biofilm growth (based on accumulated bacterial cell density, stained with crystal violet [CV], in biofilms in the growth phase) (n = 288). A₅₉₀, absorbance at 590 nm.

The reproducibility of biofilm formation in the MBEC system during the growth phase was measured as the ratio of planktonic growth to the corresponding biofilm biomass accumulation in three independent growth experiments. Our analysis demonstrated consistent and reproducible biofilm biomass accumulation and planktonic growth using a standard starting inoculum of P. aeruginosa PAO1 (Fig. 1C). The values of the mean ratio (± standard error of the mean) of planktonic growth to biofilm formation were 0.198 ± 0.002, 0.183 ± 0.002, and 0.192 ± 0.001 for experiments 1, 2, and 3, respectively. Thus, although some variations occurred between microtiter plates, as expected, variations within plates were negligible. To permit analysis across experiments, the data were normalized to values for untreated controls, as described in Materials and Methods.

Small AgNPs and aztreonam individually inhibit planktonic cell growth during antimicrobial challenge.After the growth phase, preformed biofilms were incubated in a second 96-well microtiter plate, containing different concentrations and combinations of AgNPs and/or aztreonam (challenge) (Fig. 1B). We first determined MIC values for the planktonic populations shed from the biofilms in the presence of various concentrations of either AgNPs or aztreonam alone (Table 1). The median and mode MIC values demonstrated that, when administered individually, smaller AgNPs were more effective at inhibiting P. aeruginosa planktonic growth in the challenge phase than were larger AgNPs (Table 1). Median MICs for AgNPs alone ranged from 0.234 μg/ml for the 10-nm particles to 7.50 μg/ml for the 100-nm particles. For all concentrations of aztreonam tested (2.00 μg/ml to 512 μg/ml), no planktonic growth was observed in the presence of antibiotic. These results indicated MIC values of ≤2.00 μg/ml for planktonic cells shed from biofilms treated with aztreonam in the challenge phase in this system (Table 1). Lower concentrations of aztreonam were not tested here, since our focus was on investigating inhibition of biofilm growth using these antimicrobials, as discussed below.

Aztreonam treatment alone enhances biofilm biomass accumulation in the recovery phase.After the challenge phase, treated biofilms were rinsed, moved to a third 96-well microtiter plate containing fresh medium, and incubated for 20 h to determine biofilm biomass and viability (recovery) (Fig. 1B). Biofilms treated with increasing concentrations of aztreonam alone during the challenge phase demonstrated significant increases in biomass after the 20-h recovery, with the highest concentrations of antibiotic leading to an average of ∼250% recovered biomass, compared to untreated biofilms (Fig. 2A). Biofilm viability was similar to that of untreated biofilms (∼100% with all concentrations tested) (Fig. 2A). The MBECs for P. aeruginosa biofilms following challenge with aztreonam alone were also evaluated (Table 2). These data suggest that the inhibitory effects of this antibiotic alone against preformed biofilms are limited and potentially enhance biofilm growth after antibiotic treatment has ceased.

• Open in new tab
• Download powerpoint

FIG 2

Effects of AgNPs or aztreonam alone on P. aeruginosa PAO1 biofilm biomass and viability. Values were normalized to those obtained from untreated control samples after biofilms were incubated in fresh medium in the recovery phase. (A) Biofilm biomass and viability following challenge with aztreonam (0.0 μg/ml to 512 μg/ml). (B and C) Biofilm biomass (B) and viability (C) following challenge with different sizes (10 nm to 100 nm) and concentrations (0.0 μg/ml to 10.0 μg/ml) of AgNPs.

Small AgNPs alone are more effective than larger nanoparticles in reducing biofilm biomass and viability.Treatment of P. aeruginosa PAO1 biofilms with AgNPs alone revealed that smaller AgNPs were more effective than larger nanoparticles in limiting or reducing biofilm biomass in the recovery phase, compared to untreated biofilms (Fig. 2B). Both 10-nm and 20-nm AgNPs effectively reduced biofilm biomass at concentrations of ≥0.625 μg/ml, while the 40-nm and 60-nm AgNPs reduced biofilm biomass at concentrations of ≥2.50 μg/ml. The 100-nm AgNPs demonstrated negligible effects on P. aeruginosa biofilm biomass, resulting in ∼100% biomass recovery at all concentrations tested, similar to untreated samples (Fig. 2B). Similar postchallenge trends were observed for biofilm viability (Fig. 2C). The 10-nm and 20-nm AgNPs showed the most significant reductions in biofilm viability after the challenge, with reduced viability occurring at concentrations of ≥1.25 μg/ml. The 40-nm AgNPs reduced biofilm viability at ≥2.50 μg/ml, while the 60-nm AgNPs reduced viability at ≥5.00 μg/ml. Biofilm viability following treatment with 100-nm AgNPs was consistently greater than 100% at all concentrations tested (Fig. 2C), suggesting that the larger size of these nanoparticles reduced their effectiveness as antibiofilm antimicrobials. We also calculated MBEC values for the AgNPs of various sizes (Table 2). Within the parameters tested, the largest changes in MBEC values, relative to MIC values (Table 1), were observed for the 10-nm AgNPs; the median and mode MBEC values were 16- and 32-fold higher than the corresponding MIC values, respectively.

Specific combinations of AgNPs and aztreonam inhibit biofilm biomass and viability.Next, we evaluated the recovery of P. aeruginosa biofilms after challenge with different combinations of AgNPs and aztreonam (Fig. 3). In the absence of AgNPs, the biofilm biomass was dramatically increased as the concentration of aztreonam used in the challenge phase was increased (Fig. 3A). For the 10-nm AgNPs, the first observations of significant reductions of biofilm biomass occurred at a concentration of 0.312 μg/ml, with various levels of reduction depending on the concentration of aztreonam (Fig. 3C). Increasing the 10-nm AgNP concentration to 0.625 μg/ml dramatically reduced the biofilm biomass at aztreonam concentrations of ≥8.00 μg/ml (Fig. 3D). As the AgNP concentrations were increased to 2.50 μg/ml (Fig. 3E and F), biofilms treated with 20-nm AgNPs displayed greatly reduced biomass, similar to those treated with 10-nm AgNPs (∼2% of values for untreated controls), those treated with 40-nm AgNPs displayed biomass reduced to less than ∼50%, and those treated with 60-nm AgNPs were similar to untreated cells (∼100%). This trend appeared to continue up to the highest concentration of AgNPs tested (10 μg/ml), at which biomass values for the largest nanoparticles began to fall below 100% (Fig. 3H).

• Open in new tab
• Download powerpoint

FIG 3

Effects of different AgNP-aztreonam combinations on P. aeruginosa PAO1 biofilm biomass in the recovery phase. Normalized values of percent recovery of biofilm biomass following challenge with different combinations of aztreonam (0.0 μg/ml to 512 μg/ml) and AgNP sizes (10 nm to 100 nm) at the specific AgNP concentration (0.0 μg/ml to 10.0 μg/ml) indicated in the top left corner of each panel are shown (panel A represents replicate samples treated with aztreonam only). Biofilm biomass values for treated samples were normalized to values for untreated cells (i.e., 0.0 μg/ml AgNPs and 0.0 μg/ml aztreonam). Dashed lines, 100% recovery (i.e., levels observed for untreated cells).

The viability of P. aeruginosa biofilms after challenge with different combinations of AgNPs and aztreonam followed a similar pattern overall, compared to the biomass results, with the exception of the 100-nm particles (Fig. 4). In the absence of AgNPs, biofilm viability after treatment with increasing concentrations of aztreonam recovered to ∼100% of untreated controls (Fig. 4A). At the lowest AgNP concentration of 0.156 μg/ml, the 10-nm AgNPs significantly decreased biofilm viability (from ∼100% to ∼60%) at aztreonam concentrations of ≥64.0 μg/ml, while the 100-nm AgNPs decreased biofilm viability to ∼70% at aztreonam concentrations of ≥256 μg/ml (Fig. 4B). With the 0.625-μg/ml level of the 10-nm AgNPs, biofilms were nearly nonviable at all aztreonam concentrations tested (≥2.00 μg/ml) (Fig. 4D). As the concentrations of AgNPs were increased, the 20-nm AgNPs became more effective at reducing biofilm viability, followed by the 40-nm and 60-nm AgNPs (Fig. 4D to H). An exception to this general trend was the 100-nm AgNPs, which showed variable effects on biofilm viability across both concentration gradients. It is unclear what factors may cause the variable inhibitory effects of the 100-nm AgNPs against biofilm viability, but it is clear, when these findings are combined with the corresponding biofilm biomass data for the 100-nm particles (Fig. 3), that there seems to be no overall negative effect on biofilm recovery. Together, these data suggest that, in combination with aztreonam, smaller AgNPs were more effective in reducing P. aeruginosa PAO1 biofilm biomass and viability.

• Open in new tab
• Download powerpoint

FIG 4

Effects of different AgNP-aztreonam combinations on P. aeruginosa PAO1 biofilm viability in the recovery phase. Normalized values of percent viability of biofilms following challenge with different combinations of aztreonam (0.0 μg/ml to 512 μg/ml) and AgNP sizes (10 nm to 100 nm) at the specific AgNP concentration (0.0 μg/ml to 10.0 μg/ml) indicated in the top left corner of each panel are shown (panel A represents replicate samples treated with aztreonam only). Biofilm viability values were normalized to values for untreated control samples (i.e., 0.0 μg/ml AgNPs and 0.0 μg/ml aztreonam). Dashed lines, 100% recovery (i.e., levels observed for untreated cells).

AgNPs and aztreonam exhibit antimicrobial synergy against P. aeruginosa biofilms.To directly compare the effects of combining AgNPs and aztreonam on biofilm biomass and viability, we used heat plots to visualize changes in these measures, expressed as percentages of untreated control values (Fig. 5). The heat plots indicated that smaller AgNPs had greater effects than larger AgNPs on biofilm biomass and viability at lower aztreonam concentrations. In addition, the data presented in Fig. 5 identified specific combinations of AgNPs and aztreonam concentrations that may be synergistic in preventing recovery of P. aeruginosa biofilm populations following challenge.

• Open in new tab
• Download powerpoint

FIG 5

Comparison of effects of different AgNP-aztreonam combinations on biofilm biomass (left) and biofilm viability (right) in the recovery phase, according to AgNP size (indicated on the right). Results are presented as heat plots illustrating the normalized values of percent recovery of biofilm biomass and viability after treatment with AgNPs and aztreonam. One hundred percent recovery equals levels observed for untreated control samples. Darker blue boxes, greater percent recoveries (i.e., greater biofilm biomass or viability); lighter blue boxes, poorer biofilm recovery (i.e., greater antimicrobial effect).

To confirm and to quantify this potential synergy, we used the Bliss independence model and FIC index values to determine the nature of the inhibitory effects observed when different antimicrobials were combined (35–38). We calculated the degree of antimicrobial synergy (Bliss coefficient) against P. aeruginosa biofilms for AgNPs of different sizes in combination with high (512 μg/ml), moderate (64.0 μg/ml), and low (4.00 μg/ml) concentrations of aztreonam (Fig. 6). In our calculations, positive values denote a synergistic combination of antimicrobials, and negative values describe antagonistic effects (37). The 10-nm AgNPs displayed the broadest profile of synergy over this range of AgNP and aztreonam concentrations (Fig. 6, top). The 20-nm and 40-nm AgNPs demonstrated a narrower synergy profile, with synergy revealed only at select AgNP and aztreonam concentrations. In contrast, the 60-nm and 100-nm AgNPs exhibited minimal synergy. Marked antagonism was even observed for the 100-nm AgNPs with 512 μg/ml aztreonam, with multiple values well below zero (Fig. 6, bottom left).

• Open in new tab
• Download powerpoint

FIG 6

Bliss independence model of synergy, confirming synergistic antimicrobial effects of AgNP-aztreonam combinations against P. aeruginosa biofilms. The degree of synergy was quantified using the Bliss model of synergy for recovery based on the normalized values of percent biofilm biomass after 20 h of incubation in fresh medium. For each size of AgNPs (indicated on the right), each point on the graphs represents a specific combination of AgNPs (0.0 μg/ml to 10.0 μg/ml) and aztreonam (512 μg/ml, 64.0 μg/ml, or 4.00 μg/ml). Dashed lines, no combined effects of the antimicrobials. Positive Bliss coefficient values reflect synergy and negative values represent antagonism between the combined antimicrobials.

The evaluation of synergy using the FIC index indicated that the 10-nm and 20-nm AgNPs exhibited synergistic interactions with aztreonam, while the 40-nm, 60-nm, and 100-nm AgNPs showed no interactions (Table 3). Similar to the Bliss independence model data, the 10-nm AgNPs had the broadest interactions with aztreonam to inhibit the P. aeruginosa biofilms. While some 20-nm AgNP-aztreonam combinations did result in synergistic interactions, they were not as diverse as for the 10-nm AgNPs. This complemented our analysis using the Bliss model (Fig. 6). The primary differences occurred for the interactions of aztreonam with the 40-nm, 60-nm, and 100-nm AgNPs, with the FIC indices indicating no interaction and the Bliss independence model showing some interaction.

Visualization of the antimicrobial synergy of AgNPs and aztreonam against P. aeruginosa biofilms.To visualize the effects of AgNPs and aztreonam on P. aeruginosa biofilms, we used a flow reactor to culture biofilms inside silicone tubing (4, 33, 34) (Fig. 7A). Based on the calculations from the Bliss independence model (Fig. 6) and the MBEC values in Table 2, we selected (i) concentrations of aztreonam and AgNPs that were expected to be ineffective alone and (ii) concentrations of aztreonam and 10-nm AgNPs that displayed synergy against P. aeruginosa biofilms. Consequently, we treated the biofilms with 64.0 μg/ml aztreonam alone, 10-nm or 100-nm AgNPs alone (at 0.625 μg/ml), or combinations of these treatments, and we compared the thickness of the biofilms to that of untreated control biofilms (Fig. 7B). Full-field optical coherence tomography (FFOCT) results revealed that combinations of 10-nm or 100-nm AgNPs with aztreonam, and the 10-nm AgNPs alone, significantly reduced biofilm thickness versus untreated biofilms (Fig. 7B). The combination of AgNPs with aztreonam also significantly reduced biofilm thickness in comparison with either 10-nm or 100-nm AgNPs alone (Fig. 7B). Although both sizes of AgNPs plus aztreonam reduced biofilm thickness within the silicone tubing, the combination of 10-nm AgNPs and aztreonam was the most effective. These results support the role of the smaller AgNPs in promoting synergy, as illustrated in Fig. 6.

• Open in new tab
• Download powerpoint

FIG 7

Effects of AgNP-aztreonam combinations on biofilm architecture. (A) Optical coherence tomographic slice of an untreated P. aeruginosa biofilm cultured for 72 h in silicone tubing. White arrows, biofilm; black arrowheads, areas where the biofilm detached from the inside of the silicone tubing. Black dashed lines, areas where measurements of biofilm thickness were derived. Bar, 400 μm. (B) Mean thickness (with standard deviation) of biofilms treated with various antimicrobials for 18 h (n = 60 measurements per condition). Antimicrobial treatments included 10-nm or 100-nm AgNPs at 0.625 μg/ml alone, aztreonam (Azt) at 64.0 μg/ml alone, and combinations of these treatments. *, P < 0.05 versus untreated control; **, P < 0.05 versus 10-nm AgNPs alone; ***, P < 0.05 versus 100-nm AgNPs alone.

We next used transmission electron microscopy (TEM) to explore the physical changes that occurred at the cellular level upon exposure of P. aeruginosa cells within a biofilm to AgNP and aztreonam treatments. The same silicone tubing samples described above were embedded, thin sectioned, and imaged (45) to reveal the morphology and ultrastructure of cells and the internal structure of the biofilms (Fig. 8). Micrographs of untreated biofilm controls (Fig. 8A) showed the presence of intact P. aeruginosa cells and biofilm matrix material. Biofilms treated with 64.0 μg/ml aztreonam alone or either 10-nm or 100-nm AgNPs alone (Fig. 8B, C, and D, respectively) showed no significant morphological or ultrastructural defects in comparison with untreated cells (Fig. 8A). However, biofilms treated with a combination of 10-nm AgNPs and aztreonam exhibited substantial cellular changes, including altered cellular morphology and irregularities in both the distribution of the membrane and the cytoplasmic contents of the cells, indicating that the integrity of the bacterial cell walls was likely compromised (Fig. 8E). Furthermore, 10-nm AgNPs could be detected throughout the matrix of these biofilms and were associated with cellular debris (Fig. 8G). In contrast, biofilms treated with 100-nm AgNPs and aztreonam exhibited minor cellular perturbations, compared to cells treated with 10-nm AgNPs plus aztreonam, and no 100-nm AgNPs were observed within the biofilm matrix posttreatment (Fig. 8F).

• Open in new tab
• Download powerpoint

FIG 8

Penetration of smaller AgNPs into P. aeruginosa biofilms. (A to F) Negatively stained transmission electron micrographs of P. aeruginosa PAO1 biofilms cultured in silicone tubing for 72 h and either left untreated (A) or treated for 18 h with aztreonam (64.0 μg/ml) (B), 10-nm AgNPs (0.625 μg/ml) (C), 100-nm AgNPs (0.625 μg/ml) (D), 10-nm AgNPs (0.625 μg/ml) plus aztreonam (64.0 μg/ml) (E), or 100-nm AgNPs (0.625 μg/ml) plus aztreonam (64.0 μg/ml) (F). Bars, 500 nm. (G) Inset images from panel E, highlighting the presence of 10-nm AgNPs (arrowheads). Bars, 50 nm.