Antibacterial Efficacy of AgNPs-PVP and Unprotected AgNPs
As discussed previously, S. pneumoniae and NTHi are the two most common bacteria pathogens causing OM, accounting for 58% of the total OM episodes in the US4,5. The antibacterial efficacy of AgNPs-PVP and unprotected AgNPs was thus tested using these two pathogens. The minimum inhibitory concentration (MIC) and half maximal inhibitory concentration (IC50) were used to quantify the antibacterial efficacy. MIC was obtained via broth microdilution assay, where MIC corresponds to the lowest concentration of antimicrobials that prevented bacterial growth. To better characterize the affected bacterial growth before complete eradication (i.e., MIC) was achieved, IC50 was calculated, defined as the concentration of antimicrobials that led to a stationary OD600 that was half of that without antimicrobials. For antimicrobials with the same MIC, lower IC50 indicates higher antimicrobial effect.
The antimicrobial efficacy of AgNPs against NTHi showed an average IC50 of 28.72 µM (~4.88 µg/mL) and an average MIC of 50 µM (~8.5 µg/mL) (Figure 4A). The average values of IC50 and MIC were much reduced in the presence of PVP, which became 9.45 µM (~1.61 µg/mL) and 12.5 µM (~2.13 µg/mL) respectively (Figure 4A). The greater antimicrobial efficacy of AgNPs-PVP than unprotected AgNPs was likely a result of the stabilized particulates (Figure 3) with greater surface-to-volume ratio than aggregates that led to enhanced interactions with pathogens.
The antibacterial efficacy of AgNPs-PVP was comparable to tradition antibiotics against NTHi, such as Amoxicillin (with MIC of 0.5 - 2 µg/mL), Clarithromycin (with MIC of 2 - 8 µg/mL), and Azithromycin (with MIC of 0.25 - 2 µg/mL)62. Contrary to those small-molecule antibiotics, efficacy of AgNPs and AgNPs-PVP againstS. pneumoniae was better than that against NTHi (Figure 4B), with IC50 values of 17.88 µM (~2.99 µg/mL) and 4.14 µM (~0.70 µg/mL) for AgNPs and AgNPs-PVP respective and MIC values of 25 µM (~4.18 µg/mL) and 6.25 µM (~1.04 µg/mL) for AgNPs and AgNPs-PVP respective. The stronger antimicrobial effects of AgNPs and AgNPs-PVP against S. pneumoniae than that against NTHi could be due to their gram types or the known production of H2O2 by S. pneumoniae (at levels around 0.1 – 0.71 mM as a mechanism for competitive survival during coinfections)63. To better understand this differential efficacy of AgNPs-PVP, Streptococcus mutans (S. mutans) , a gram-positive pathogen (same as S. pneumoniae ) with much lower activity of H2O2 production (at levels around 0 - 0.06 mM64,65) was tested.
S. mutans is one of the microorganisms inhabiting the oral cavity, which has been studied for its etiology of dental caries and infective endocarditis66. Interestingly, the colonization of S. mutans in mouth has been shown to be correlated with the colonization of S. pneumonia in the nasopharynx67, thus making S. mutans a pathogen of interest for OM treatment. Indeed, the MIC values of AgNPs and AgNPs-PVP against S. mutans were 50 µM (~8.35 µg/mL) and 12.5 µM (~2.09 µg/mL) respectively, which were comparable to NTHi and greater than S. pneumoniae (Figure 4C). Similarly, the IC50 value of AgNPs and AgNPs-PVP against S. mutans were 32.03 µM (~5.35 µg/mL) comparable to that of NTHi. The IC50 value of AgNPs-PVP, 4.87 µM (~0.81 µg/mL), was smaller than that of NTHi (9.45 µM (~1.58 µg/mL)) and close to that ofS. pneumoniae (4.14 µM (~0.70 µg/mL)), which could be explained by the bacteriostatic effect of low levels of H2O2 on S. mutans 68. Taken together, the MIC and IC50 values of AgNPs and AgNPs-PVP against S. mutans confirmed that their greater efficacy against S. pneumoniae was likely a result of the H2O2-producing capability and not gram types, as gram-positive S. mutans and gram-negative NTHi demonstrated comparable values.